Control of acidosis

ABSTRACT

The present invention relates to a vaccine for the prevention of lactic acidosis in a vertebrate, said vaccine comprising at least one isolated microorganism, or fragment or fragments thereof, wherein said microorganism is capable of producing lactic acid within the gut of said vertebrate, and wherein said microorganism is selected from the group consisting of:  Clostridium -like species,  Prevotella -like species,  Bacteroides -like species,  Enterococcus -like species,  Selenomonas  species, non-dextran slime producing  Streptococcus  species and non-slime producing lactic acid bacterial isolates.

TECHNICAL FIELD

The present invention relates to the identification of a series ofmicroorganisms involved in the development of acidosis, together withthe treatment and/or prevention and/or detection of acidosis invertebrates, in particular, vaccines, compositions and methods for thetreatment and/or prevention and/or detection of acidosis in vertebrates.

BACKGROUND ART

The over production of acid in the gut of an animal by microorganismscan cause fermentative acidosis, wherein is defined as a condition ofabnormally high acidity in the gut that can lead to local and systemicacidosis, damage to the integrity of the gut wall and increasedpathogenicity of gut bacteria and parasites.

The introduction of starch, sugars or oligosaccharides into the rumen ofruminant animals and the hind gut (caecum and colon) of ruminant andnon-ruminant animals including humans, leads to rapid fermentation andproduction of volatile fatty acids (VFA). As the rate of VFA productionexceeds their rate of removal, the pH may fall below 6.0, such thatlactobilli take over, fermenting the starch to produce more lactic acidand creating an even lower pH (e.g. below 5.5). This is the scenariooften presented to describe the sequential reactions in the rumen or thehind gut that lead to fermentative acidosis. The conditions of acidosiscan be acute, posing an immediate life-threatening situation, or chronic(sub-acute), resulting in reduction in both feed intake and weight gain.There are also numerous disease conditions that can develop as secondaryand tertiary consequences of acid accumulation in the gut.

Among the several methods for reducing the risk of acidosis, the use ofantibiotic feed additives such as virginiamycin or certain ionophoreshave been relatively effective. However, under certain feedingconditions addition of virginiamycin has not always reduced the risk ofacidosis (Godfrey et al., 1995; Courtney and Seirer, 1996; Thomiley etal., 1998).

Accordingly, there is a need to provide alternative means of controllingfermentative acidosis.

The main bacterial species and strains identified in the presentinvention, Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates, have not previously beenconsidered to be important organisms in the development of fermentativeacidosis. In fact, in the case of Selenomonas ruminantium it should benoted that the isolate selected by Leedle (1970) was identified on thebasis of its ability to utilise lactic acid, rather than produce it andis therefore very different to the isolates that form part of thepresent invention.

Therefore, the present invention has identified a series ofmicroorganisms responsible for the development of acidosis and describesvaccines, compositions and methods for the treatment and/or preventionand/or detection of acidosis in vertebrates.

DETAILED DESCRIPTION OF THE INVENTION

1. Vaccine/Pharmaceutical Compositions for Control of Acidosis

According to a first embodiment of the invention, there is provided avaccine for the prevention of lactic acidosis in a vertebrate, saidvaccine comprising at least one isolated microorganism, or fragment orfragments thereof, wherein said microorganism is capable of producinglactic acid within the gut of said vertebrate, and wherein saidmicroorganism is selected from the group consisting of: Clostridium-likespecies, Prevotella-like species, Bacteroides-like species,Enterococcus-like species, Selenomonas species, non-dextran slimeproducing Streptococcus species and non-slime producing lactic acidbacterial isolates.

Typically, in the vaccine of the present invention, the microorganismforms part of the normal gut flora of a vertebrate. More typically, themicroorganism is involved in the aetiology of fermentative lacticacidosis in vertebrates. Still more typically, the microorganism isselected from the group consisting of: Streptococcus equinus,Clostridium-like vitulinus, Selenomonas ruminantium, Prevotella-likespecies, Bacteroides-like species, Enterococcus-like species,Streptococcus bovis SbR1 and non-slime producing lactic acid bacterialisolates LAB02, LAB06 and LAB08. Yet still more typically, themicroorganism is selected from the group consisting of: Streptococcusbovis (SbR1), Streptococcus equinus (SER1); Streptococcus equinus(SER2); Selenomonas ruminantium (SRR1); Selenomonas ruminantium (SRR3);Clostridium-like vitulinus (LVR3); Clostridium-like vitulinus (LVR4).

Yet even still more typically, the microorganism is one of the followingstrains deposited with the Australian Government Analytical Laboratories(AGAL) of 1 Suakin Street, Pymble, New South Wales, Australia, on 24Jun. 1999 and given accession numbers as outlined in the followingtable:

Microorganism Accession number Streptococcus bovis (SbR1 A2): NM99/04455Streptococcus equinus (SER1): NM99/04456 Streptococcus equinus (SER2):NM99/04457 Selenomonas ruminantium (SRR1) NM99/04458 Selenomonasruminantium (SRR3) NM99/04460 Clostridium-like vitulinus (LVR3)NM99/04461 Clostridium-like vitulinus (LVR4) NM99/04462

Alternatively, the microorganism is one of the following strainsdeposited with the Australian Government Analytical Laboratories (AGAL)of 1 Suakin Street, Pymble, New South Wales, Australia, on 29 Jun. 2000and given accession numbers as outlined in the following table:

Accession Lab. code Description No. LAB 01/07-3 Prevotella-like, shortrods arranged in short NM00/12630 chains and filaments. Gram positiveLAB 02/11-2 non-slime producing lactic acid bacterial NM00/12631isolate, short straight rods with round ends, also long thin rods LAB03/D35 Prevotella-like, predominantly rods in NM00/12632 filaments.Gram+ LAB 04/D37 Non-dextran slime producing Streptococcus NM00/12633isolate predominantly large cocci. occurring mainly in pairs and singly(larger than the S. bovis isolates SB R1 or Sb5) LAB 05/D23Bacteroides-like predominantly small NM00/12634 cocci < than 1 μm indiameter mainly as diplococci: all Gram positive LAB 06/D29 Non-slimeproducing lactic acid bacterial NM00/12635 isolate. long > than 5 μmthick rods, in singles and short chains LAB 07/H1 Bacteroides-like,predominantly short rods NM00/12636 forming filaments. Morphologicallysimilar to those isolated from pigs (LAB01/07-3) LAB 08/H15 Non-slimeproducing lactic acid bacterial NM00/12637 isolate. an endospore-formingbacteria. rod shaped. straight with a terminal spores. The spores arecylindrical. oval and round in shape, Gram positive

Clostridium-like vitulinus (LVR3) and Clostridium-like vitulinus (LVR4)are new isolates. In relation to this, the 16S rRNA gene sequencing dataas provided in the sequence listing (SEQ ID NOS: 1–7) of the presentinvention makes it clear that the organism is not a Lactobacillus andthat it is closer to a Clostridium. However, it is not a spore-formingbacterium and cannot be classified as a member of the genus Clostridiumper se. Consequently, it is necessary to name a new genus to cover thespecies vitulinus, and throughout the present specification, theorganism is referred to as Clostridium-like vitulinus.

Further, in Streptococcus bovis (SbR1) (NM99/04455) strain of thepresent invention is distinct from other Streptococcus bovis strains,such as Streptococcus bovis (Sb5) (N94/8225), deposited with theAustralian Government Analytical Laboratories (AGAL) of 1 Suakin Street,Pymble, New South Wales, Australia, on 8 Mar. 1994 strain, on the basisof a number of differentiating factors. For instance, the Streptococcusbovis (SbR1) (NM99/04455) strain produces far less dextran exudativeslime material than for example, Streptococcus bovis Sb5. Lack ofdextran slime formation has important implications for the antigenicityand for vaccine production since the slime makes harvesting of the cellsconsiderably more difficult. For example, in the case of Streptococcusisolates, dextran (slime) characteristics may be examined bycentrifugation, and the absence of a bacterial “pellet” followingcentrifugation indicates a dextran type slime. One of the aims of thepresent invention was the selection for S. bovis bacteria that did notproduce dextran slime.

Typically, the vertebrate is a monogastric, herbivore or ruminant animalor human subject. Even more typically, the vertebrate is selected fromthe group consisting of human, non-human primate, murine, bovine, ovine,equine, porcine, caprine, leporine, avian, feline and canine. Moretypically, the vertebrate is selected from the group consisting ofhuman, ovine, camelids, porcine, bovine, equine or canine.

Typically, the vaccine comprises live or dead intact cells of at leastone of the microorganisms as defined in accordance with the firstembodiment of the invention. More typically, the vaccine comprises celllysate from at least one fermentative lactic acid producingmicroorganisms defined in the first embodiment of the invention. Evenmore typically, the vaccine comprises crude antigen mixture or purifiedantigen or antigens from at least one fermentative lactic acid producingmicroorganism defined in the first embodiment of the invention. Stillmore typically, the vaccine comprises outer membrane and associatedproteins of at least one of the microorganisms defined in the firstembodiment of the invention.

Typically, the fragment or fragments of the microorganism is present inthe vaccine as an immunogenic polypeptide, glycopeptide or the like.

Typically, the vaccine comprises both live or dead intact cells of atleast one of the microorganisms as defined in accordance with the firstembodiment of the invention, together with outer membrane and associatedproteins of at least one of these microorganisms, and/or a fragment(s)of at least one of these microorganism present as an immunogenicpolypeptide, glycopeptide or the like.

Still more typically, the vaccine may be comprised of a combination ofone of the following: live or dead intact cells of at least one of themicroorganisms as defined in accordance with the first embodiment of theinvention, outer membrane and associated proteins of at least one ofthese microorganisms, both dead intact cells of at least one of thesemicroorganisms, together with outer membrane and associated proteins ofat least one of the microorganisms of the invention, and/or afragment(s) of the microorganism present as an immunogenic polypeptide,glycopeptide or the like, wherein the live or dead intact cells or outermembrane and associated proteins or immunogenic polypeptide,glycopeptide or the like, are derived from at least two microorganismsof the invention.

Typically, the vaccine is formulated for administration viaintramuscular, subcutaneous, topical or other parenteral route. Ingeneral, the microorganisms of the present invention are commensal innature, with a presence in the gut. Thus, oral administration isgenerally not an effective route of vaccination, and as a consequence,administration via an intramuscular, subcutaneous topical or otherparenteral route is preferred.

Typically, the vaccine may also include cytokines, such as: G-CSF,GM-CSF, interleukins or tumour necrosis factor alpha, used singly or incombination.

Typically, the vaccine may comprise a combination of two or more of themicroorganisms outlined in accordance with the first embodiment of theinvention.

According to a second embodiment of the invention, there is provided apharmaceutical composition for the prevention of lactic acidosis in avertebrate comprising at least one isolated microorganism capable ofproducing lactic acid within the gut of a vertebrate, or fragment orfragments thereof, wherein said microorganism is selected from the groupconsisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates, together with apharmaceutically acceptable carrier, adjuvants and/or diluent.

Typically, in the pharmaceutical composition of the present invention,the microorganism forms part of the normal gut flora or a vertebrate.More typically, the microorganism is involved in the aetiology offermentative lactic acidosis in vertebrates. Still more typically, themicroorganism is selected from the group consisting of: Streptococcusequinus, Clostridium-like vitulinus, Selenomonas ruminantium,Prevotella-like species, Bacteroides-like, Enterococcus-like species,Streptococcus bovis SbR1 and non-slime producing lactic acid bacterialisolates LAB02, LAB06 and LAB08. Yet still more typically, themicroorganism is selected from the group consisting of: Streptococcusbovis (SbR1), Streptococcus equinus (SER1); Streptococcus equinus(SER2); Selenomonas ruminantium (SRR1); Selenomonas ruminantium (SRR3);Clostridium-like vitulinus (LVR3); Clostridium-like vitulinus (LVR4),Prevotella-like isolates LAB01 and LAB03, Bacteroides-like isolatesLAB07 Enterococcus-like isolate LAB05, Streptococcus bovis SbR1,non-dextran slime producing Streptococcus isolate LAB04 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08.

More typically, the microorganism(s) within the pharmaceuticalcomposition is selected from the group consisting of: Streptococcusbovis (SbR1) (NM99/04455), Streptococcus equinus (SER1) (NM99/04456);Streptococcus equinus (SER2) (NM99/04457); Selenomonas ruminantium(SRR1) (NM99/04458); Selenomonas ruminantium (SSR3) (NM99/04460);Clostridium-like vitulinus (LVR3) (NM99/04461); Clostridium-likevitulinus (LVR4) (NM99/04462), Prevotella-like isolates LAB01(NM00/12630) and LAB03 (NM00/12632), Bacteroides-like isolates LAB07(NM00/12636), Enterococcus-like isolate LAB05 (NM00/12634),Streptococcus bovis (SbR1), non-dextran slime producing Streptococcusisolate LAB04 (NM00/12633) and non-slime producing lactic acid bacterialisolates LAB02 (NM00/12631), LAB06 (NM00/12635) and LAB08 (NM00/12637).

Typically, the microorganism(s) within the pharmaceutical composition isprovided as live cells, attenuated cells, killed whole cells, celllysate, crude antigen mixture or purified antigen or antigen from themicroorganism. More typically, the microorganism, and/or fragment orfragments thereof, is present in the pharmaceutical composition as outermembrane and associated proteins of the microorganism. Even moretypically, the microorganism or glycopeptide, or the like. Yet stillmore typically, the pharmaceutical composition further comprises atleast one cytokine, such as: G-CSF, GM-CSF, interleukin or tumornecrosis factor alpha, used singly or in combination.

Typically, the pharmaceutical composition may comprise a combination oftwo or more of the microorganisms defined the second embodiment of theinvention.

Typically, the microorganism present in the pharmaceutical compositionmay exist as a monoculture of at least one microorganism defined in thesecond embodiment of the invention, or may be present as a mixed culturewith other microorganisms, wherein the predominant microorganism(s) isthat defined in the second embodiment of the invention.

Typically, the pharmaceutical composition in accordance with the secondembodiment of the invention may also include an adjuvant. Moretypically, the adjuvant is selected from the group consisting of:Freunds Complete/Incomplete Adjuvant, Montenide Macrol Adjuvant,Phosphate Buffered Saline and Mannan oil emulsions, saponins (QuiLA)dextran (dextran sulphate, DEAE-Dextran), aluminum compounds (ImjectAlum), N-acetylglucosamiyl-N-acetylmuramyl-L-alanyl-D-isoglutamine(Gerbu adjuvant). More typically, the adjuvant is selected from thegroup as described in the Vaccine 1995, vol 13, p 1203; 1993 vol 11 p293; and 1992 vol 10 p 427, the disclosures of which are incorporatedherein by reference.

2. Methods for Control of Acidosis

According to a third embodiment of the invention, there is provided amethod for inducing an immune response against lactic acidosis in avertebrate, said method comprising administering to said vertebrate animmunologically effective amount of the vaccine in accordance with thefirst embodiment of the invention, or a pharmaceutical composition inaccordance with the second embodiment of the invention.

According to a fourth embodiment of the invention, there is provided thevaccine as defined in accordance with the first embodiment of theinvention, or a pharmaceutical composition as defined in accordance withthe second embodiment of the invention, when used in inducing an immuneresponse against lactic acidosis in a vertebrate.

According to a fifth embodiment of the invention, there is provided theuse of at least one isolated microorganism capable of producing lacticacid within the gut of a vertebrate, or fragment or fragments thereof,wherein said microorganism is selected from the group consisting of:Clostridium-like species, Prevotella-like species, Bacteroides-likespecies, Enterococcus-like species, Selenomonas species, non-dextranslime producing Streptococcus species and non-slime producing lacticacid bacterial isolates, or a pharmaceutical composition as defined inaccordance with the second embodiment of the invention, in thepreparation of a vaccine for the inducing an immune response againstlactic acidosis in vertebrate.

Still more typically, the microorganism is selected from the groupconsisting of: Streptococcus equinus, Clostridium-like vitulinus,Selenomonas ruminantium, Prevotella-like species, Bacteroides-likespecies, Enterococcus-like species, Streptococcus bovis SbR1 andnon-slime producing lactic acid bacterial isolates LAB02, LAB06 andLAB08. Yet still more typically, the microorganism is selected from thegroup consisting of: Streptococcus bovis (SbR1), Streptococcus equinus(SER1); Streptococcus equinus (SER2); Selenomonas ruminantium (SRR1);Selenomonas ruminantium (SSR3); Clostridium-like vitulinus (LVR3);Clostridium-like vitulinus (LVR4), Prevotella-like isolates LAB01 andLAB05, Bacteroides-like isolates LAB07, Enterococcus-like isolate LAB05,Streptococcus bovis SbR1, non-dextran slime producing Streptococcusisolate LAB04 and non-slime producing lactic acid bacterial isolatesLAB02, LAB06 and LAB08.

Yet still more typically, the microorganism(s) is selected from thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462),Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like isolate LAB07 (NM00/12636), Enterococcus-like isolateLAB05 (NM00/12634), Streptococcus bovis (SbR1), non-dextran slimeproducing Streptococcus isolate LAB04 (NM00/12633) and non-slimeproducing lactic acid bacterial isolates LAB02 (NM00/12631), LAB06(NM00/12635) and LAB08 (NM00/12637).

Typically, the vaccine or pharmaceutical composition administered inaccordance with the third, fourth or fifth embodiments of the invention,may also be simultaneously or sequentially administered with cytokines,such as: G-CSF, GM-CSF, interleukins or tumour necrosis factor.Cytokines can also be combined with adjuvants to enhance the immuneresponse.

According to a sixth embodiment of the invention, there is provided amethod for the treatment and/or prophylaxis of lactic acidosis in avertebrate in need of said treatment and/or prophylaxis, wherein saidmethod comprises administering to said vertebrate a therapeuticallyeffective amount of the vaccine in accordance with the first embodimentof the invention, or a pharmaceutical composition in accordance with thesecond embodiment of the invention.

According to a seventh embodiment of the invention, there is providedthe vaccine as defined in accordance with the first embodiment of theinvention, or the pharmaceutical composition as defined in accordancewith the second embodiment of the invention, when used in the treatmentand/or prophylaxis of lactic acidosis in a vertebrate in need of saidtreatment and/or prophylaxis.

According to an eighth embodiment of the invention, there is providedthe use of at least one isolated microorganism capable of producinglactic acid within the gut of a vertebrate, or fragment or fragmentsthereof, wherein said microorganism is selected from the groupconsisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates, in the preparation of amedicament for the treatment and/or prophylaxis of disease in avertebrate in need of said treatment and/or prophylaxis.

Typically, the microorganism(s) used in accordance with the eighthembodiment of the invention are selected from the group consisting of:Streptococcus equinus, Clostridium-like vitulinus, Selenomonasruminantium, Prevotella-like species, Bacteroides-like species,Enterococcus-like species, Streptococcus bovis SbR1 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08. Evenmore typically, the microorganism is selected from the group consistingof: Streptococcus bovis (SbR1), Streptococcus equinus (SER1);Streptococcus equinus (SER2); Selenomonas ruminantium (SRR1);Selenomonas ruminantium (SRR3); Clostridium-like vitulinus (LVR3);Clostridium-like vitulinus (LVR4), Prevotella-like isolates LAB01 andLAB03, Bacteroides-like isolates LAB07, Enterococcus-like isolate LAB05,Streptococcus bovis SbR1, non-dextran slime producing Streptococcusisolate LAB04 and non-slime producing lactic acid beneath isolatesLAB02, LAB06 and LAB08.

Yet still more typically, the microorganism(s) is selected from thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SSR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3) (NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462),Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like isolate LAB07 (NM00/12636). Enterococcus-like isolateLAB05 (NM00/12634), Streptococcus bovis (SbR1), non-dextran slimeproducing Streptococcus isolate LAB04 (NM00/12633) and non-slimeproducing lactic acid bacterial isolates LAB02 (NM00/12631). LAB06(NM00/12635) and LAB08 (NM00/12637).

Typically, the method in accordance with the third or sixth embodimentof the invention, the vaccine of pharmaceutical composition inaccordance with the fourth or seventh embodiment of the invention, orthe use in accordance with the fifth or eighth embodiment of theinvention, further comprises administering at least one cytokine, suchas: G-CSF, GM-CSF, interleukins or tumour necrosis factor.

Typically, the method in accordance with the third or sixth embodimentsof the invention, the variance or pharmaceutical composition inaccordance with the fourth or seventh embodiment of the invention, orthe use in accordance with the fifth or eighth embodiment of theinvention, further comprises administering an active agent to thevertebrate to assist in the treatment and/or prophylaxis of lacticacidosis in the vertebrate in need of said treatment and/or prophylaxis.

Typically, in the method in accordance with the third or sixthembodiment of the invention, the vaccine or pharmaceutical compositionin accordance with the fourth or seventh embodiment of the invention, orthe use in accordance with the fifth or eighth embodiment of theinvention, the active agent is selected from the group consisting of:antibiotics, enzyme preparations, clay preparations, compounds whichslow the digesta flow, prebiotics and probiotics.

According to a ninth embodiment of the invention, there is provided amethod for the treatment and/or prophylaxis of lactic acidosis in avertebrate in need of said treatment and/or prophylaxis, wherein saidmethod comprises administering to said vertebrate a therapeuticallyeffective amount of an active agent capable of preventing or controllinglactic acid accumulation in the gut of a vertebrate, and wherein saidlactic acid is produced by at least one microorganism selected from thegroup consisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates.

According to a tenth embodiment of the invention, there is provided anactive agent capable of preventing or controlling lactic acidaccumulation in the gut of a vertebrate, when used in the treatmentand/or prophylaxis of lactic acidosis in a vertebrate in need of saidtreatment and/or prophylaxis, wherein said lactic acid is produced by atleast one of the isolated microorganisms selected from the groupconsisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates.

According to an eleventh embodiment of the invention, there is provideduse of an active agent capable of preventing or controlling lactic acidaccumulation in the gut of a vertebrate, in the preparation of amedicament for the treatment and/or prophylaxis of lactic acidosis in avertebrate in need of said treatment and/or prophylaxis, wherein saidlactic acid is produced by at least one of the isolated microorganismsselected from the group consisting of: Clostridium-like species,Prevotella-like species, Bacteroides-like species, Enterococcus-likespecies, Selenomonas species, non-dextran slime producing Streptococcusspecies and non-slime producing lactic acid bacterial isolates.

Typically, in the method, active agent or use in accordance with theninth, tenth or eleventh embodiments of the invention, the microorganismis selected from the group consisting of: Streptococcus equinus,Clostridium-like vitulinus, Selenomonas ruminantium, Prevotella-likespecies, Bacteroides-like species, Enterococcus-like species,Streptococcus bovis SbR1 and non-slime producing lactic acid bacterialisolates LAB02, LAB06 and LAB08. Even more typically, the microorganismis selected from the group consisting of: Streptococcus bovis (SbR1),Streptococcus equinus (SER1); Streptococcus equinus (SER2); Selenomonasruminantium (SRR1); Selenomonas ruminantium (SRR3); Clostridium-likevitulinus (LVR3); Clostridium-like vitulinus (LVR4), Prevatella-likeisolates LAB01 and LAB03, Bacteroides-like isolates LAB07,Enterococcus-like isolate LAB05, Streptococcus bovis SbR1, non-dextranslime producing Streptococcus isolate LAB04 and non-slime producinglactic acid bacterial isolates LAB02, LAB06 and LAB08.

Yet still more typically, the microorganism(s) is selected from thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462),Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like isolate LAB07 (NM00/12636), Enterococcus-like isolateLAB05 (NM00/12634), Streptococcus bovis (SbR1), non-dextran slimeproducing Streptococcus isolate LAB094 (NM00/12633) and non-slimeproducing lactic acid bacterial isolates LAB02 (NM00/12631), LAB06(NM00/12635) and LAB08 (NM00/12637).

Typically, in the method, active agent or use in accordance with theninth, tenth or eleventh embodiments of the invention, the active agentis used in conjunction with a vaccine according to the first orseventeenth embodiment of the invention.

Typically, in the method, active agent or use in accordance with theninth, tenth or eleventh embodiments of the invention, the active agentis selected from the group consisting of: antibiotics, enzymepreparations, clay preparations, compounds which slow the digestionflow, prebiotics and probiotics.

The following relates to any one of the third through to eleventhembodiments of the invention.

Typically, the antibiotic is active against gram-positive lactic acidproducing microorganisms.

Typically, the enzyme preparation is active against lactic acidproducing gram-negative bacteria.

Typically, the clay preparation is active against lactic acid producingGram-negative or Gram positive bacteria.

Typically, the compounds which slow digesta flow rate are indirectlyactive against lactic acid producing gram-negative bacteria. Moretypically, the compounds which slow digesta flow rate are typicallyselected from the group consisting of biologically active peptides(BAP), compounds active on the automatic nervous system. 5HTagonists/antagonists, motilin antagonists, NO promoters, and dopamine.

Typically, the probiotic preparation include bacteria selected from thegroup consisting of: Megasphera, Veillenolla, Selenomonas,Propionibacterium, Anaerovibro and Peptococcus. More typically, theprobiotic preparations include yeast and mycelial preparations capableof utilising lactic acid, and converting lactic acid to volatile fattyacids and other end products.

Typically, the active agent is active against at least one of theisolated microorganisms as defined in accordance with the firstembodiment of the invention.

Typically, the antibiotic is an antibiotic active against lactic acidproducing bacteria that can be selected from any listing of antibioticcompounds such as available in text books, and reports such as theReport of the Joint Expert Advisory Committee on Antibiotic Resistance(JETACAR, 1999), the disclosure of which is incorporated herein byreference.

More typically, the antibiotic is selected from the group consisting of:Acyclovir (Zovirax), Amantadine (Symmetrel), Amikacin )generic),Gentamicin (generic), Tobramycin (generic), Amoxicillin (generic),Amoxicillin/Clavulanate (Augmentin), Amphoetericin B (Fungizone),Ampicillin (generic), Atovaquon (Mepron), Cefazolin (generic), Cefepime(Maxipime), Cefotaxime (Claforan), Cefuroxime (Zinacef),Chloramphienicol (generic), Clotrimazole (Mycelex), Ciprofloxacin(Cipro), Clarithromycin (Biaxin), Dicloxacillin (generic), Doxycycline(generic), Erythromycin lactobionate and other salts Fluconazole(Diflucan), F scarnet (Foscavir), Ganciclovir, (Cytovene, DHPG),Imipenem/Cilastatin (Primaxin), Ketoconazole (generic), Metronidazole(Flagyl), Nitrofurantoin Nystatin (generic) fluconazole, oramphotericin, Penicillin T (generic) (sodium or potassium salt),Pentamidine (generic), Piperazillin/Tazobactam (Zosyn), Rifampin(Rifadin), Ticarcillin/Clavulanate (timentin), TrimethoprinSulfamethoxazole, Vancomycin (generic), and any combination thereof.

More typically, the antibiotics may be used in combination with anyantibiotic agents active against lactic acid producing bacteria such asStreptococcus spp. Clostridium-like and Lactobacillus spp. Still moretypically, the antibiotic agents active against gram-positive lacticacid producing bacteria may be selected from the group consisting of:glycopeptide antibiotics, more typically, ardacin, avoparcin,telcoplanin or vancomycin; glycolipid antibiotics, more typicallyflavomycin (bambermycin); Streptogramin antibiotics, more typicallyvirginiamycin; polypeptide antibiotics, more typically bacitracin zinc,bacitracin methylene disalicylate, virginiamycin S or polymixins (B &E); macrolide antibiotics, more typically tylosin, spiramycin,virginiamycin M. josamycin, spectinomycin or erythromycin; orsulfur-containing peptide antibiotics, more typically thiopeptone,thiopeptin, sulfomycin, thiostrepton, sporangiomycin, siomycin ortaitomycin; lincosamide antibiotics, more typically lincomycin orclindamycin; or pleuromutilins tiamulin; or nitrofuran antibiotics, moretypically nitrofurantoin, nitrofurazone or furazolidone; tetracyclineantibiotics, more typically clortetracycline or oxytetracycline;doxycycline, minocycline, penicillin antibiotics, more typicallypenicillinase-resistant penicillins, such as oxacillin or methicillin,penicillin V or amplicillin; polythiazole antibodies, more typicallynosiheptide; or ionophore antibiotics, more typically lasalocid,tetronasin, naracin or salinomycin; or, novoiocin sodium, bottromycintartrate; streptogramin antibiotics, more typically,quinupristin/dalfopristin (RP 59500; Syneroid) or streptogramincombinations [quinupristidn/dalfopristin (RP 59500; Syneroid)],everninomycin derivatives (SCH 27899), oxazolidinones (U-100572,U-100766), fluoroquinolone antibiotics, more typically, ciprofloxacin,oxfloxacin, clinafloxacin, DU 6859a, grepafloxacin, levofloxacin,sparfloxacin or eprofloxacin, trovafloxacin; beta-lactam antibiotics;nitrovin (payzone), enramycin, mupiricin, magainin antibiotics,chloramphenicols and related compounds, including fluorphenicolthiamphenicol, and any combination thereof.

Typically, the antibiotics active agent against lactic acid producingmiroorganisms may be used in combination with the vaccine in accordancewith the first or seventeenth embodiments of the invention.

Typically, the antibiotics active against gram-positive lactic acidproducing microorganisms may be used in conjunction with the vaccine inaccordance with the first or seventh embodiments of the invention. Forexample animals may be immunised against Selenomonas type bacteria andfed diets containing the antibiotic virginiamycin active againstStreptococcus spp. and lactobacilli of lactic acid producing bacteria.

Typically, vaccines against Gram positive lactic acid producing bacteriacan be used in combination with the vaccine in accordance, with thefirst or seventeenth embodiments of the invention. For example, animalsmay be immunised against the Gram negative Selenomonas type bacteria andagainst Streptococcus spp. and Clostridium-like lactic acid producingbacteria.

Typically, vaccines against Gram positive lactic acid producing bacteriamay also be used in combination with vaccines against Gram negativelactic acid producing bacteria, and these vaccines can also be used inconjunction with antibiotic compounds active against lactic acidbacteria.

Typically, the enzyme preparation is active against lactic acidproducing gram-negative bacteria. More typically, enzyme preparationsare designed to reduce the passage of fermentable carbohydrate to thehind gut through improving the digestion and absorption in the intestineof starches, disaccharides, oligosaccharides, non-starchpolysaccharides, protein starch complexes and any polysaacharide whichis incompletely digested in the intestine, but which is readilyfermentable in the hind gut.

Typically, preferred enzymes for the break down of non-starchpolysaccharides and starches include the following: glyconasesincluding: amylase, maltase, invertase, α-glucosidases, emulsin, andamyloglucosidase; β-glucanases β-glucanase, xylanase; enzymes whichbreak down galactosides of the raffinosse series and otherα-galactosides including α-galactosidase, enzymes which break down theproteins forming part of the matrix surrounding starches, sugars andnon-starch carbohydrates in plant material, including: pepsin, trypsin,trypsinogen, chymotrypsin and natural and synthetic porteolytic enzymesof chemical or microbial origin, enzymes which depolymerise non-starchpolysaccharides including: arabinoxylans and β-glucans, and enzymesactive in the break down of cellulose, including: cellulase, enzymesactive in the break down of colloidal polysaccharides, pecticsubstances, which include: galactouronans, galactan and arabinans, aswell as the neutral polysaccharides such as xyloglucans andgalactomannans and other non-starch polysaccharides such as:rhamnogalactouronan with arabinose and galactose, arabinogalactan,glucan, xyloglucan, galactouronan with arabinose and uronan witharabinose. These enzymes can be used individually or in combination.

Typically, the enzyme preparation active against lactic acidaccumulation from gram-negative lactic acid producing microorganisms maybe used in conjunction with the vaccine in accordance with the first orseventeenth embodiments of the invention.

Typically, the clay preparation is active against lactic acid producingGram-negative or Gram positive bacteria. More typically, claypreparations are designed to reduce the rate of fermentation and bindsspecific ions in a way which reduces the adverse effects of rapidfermentation of starch and other soluble carbohydrates in thegastrointestinal tract.

Typically, preferred clays for reducing the rate of fermentation and theosmotic effects of rapid fermentation within the gut include: kaolinite,bentonite, montmorrilonite, illite, clinolipolite, heulandite,palygorsite, saponite, smectite, chrysotile, lizaridite, talc,pyrophhyllite, vermiculite, beidellite, halloysite or zeolite types ofclay, and these can be activated by a wide range of ions includingsodium, calcium, potassium and mixtures of these and other ions. Theseclays can be used individually or in combination.

Typically, the clay preparation active against lactic acid accumulationfrom gram-negative lactic acid producing microorganisms may be used inconjunction with the vaccine in accordance with the first or seventeenthembodiments of the invention.

Typically, the compounds which slow digesta flow rate are indirectlyactive against lactic acid producing gram-negative bacteria. Moretypically, by administering compounds which slow digesta flow rate,intestinal digestion and absorption are increased, reducing the amountof fermentable substrate passing to the hind gut.

Generally, preferred agents to slow the flow of digestion includebiologically active peptides (BAP) in a form which will reach theduodenum, and are active in modulating the activity of the digestivetrack, gastric emptying and the rate of passage through the intestine.More typically, these biologically active peptides include opioidpeptides. Compounds active on the autonomic nervous system (eg atropineand atropine-like compounds) may affect digesta flow and have similareffects. Compounds such as 5HT agonists/antagonists, motilinantagonists, NO promoters, dopamine agonists may also be used.

Whilst a range of proteins potentially produce opioid peptides onhydrolisation, the β-casomorphins, which can be derived from β-caseinincreasing casein during β-casein digestion, and particularly active.

Even more typically, the biologically active peptides includecholecystokinin (CCK), the M1 fraction of virginiamycin and the analogueof virginiamycin fraction M1 compound L-156. These biologically activepeptides can be used individually or in combination.

It has traditionally been assumed that the nutritional benefits ofproteins are only related to the essential amino acids supplied to theanimal during digestion and absorption. However through the supply ofbiologically active peptides and the production of naturally occurringopioid peptides, the rate of digesta passage is reduced and this resultsin more efficient intestinal digestion and less fermentable substratepassing to the hind gut which can contribute to acidic gut syndrome.

Practical methods of supplying biologically active opioid peptides isthrough dietary supplementation with proteins such as casein and bloodmeal. For ruminant animals the best results are obtained throughprotection of the protein against rumen degradation by polymer coatingtechnology, slow-release capsules, or through formaldehyde treatment.

Typically, the compounds which slow digesta rate and thereby activeagainst lactic acid accumulation from gram-negative lactic acidproducing microorganisms may be used in conjunction with the vaccine inaccordance with the first or seventeenth embodiment of the invention.

Typically, probiotic agents are also active against lactic acidproducing gram-negative bacteria. More typically, the probiotic reduceslactic acid accumulation from at least one of the microorganisms asdefined in accordance with the first embodiment of the invention by:formation of alternative end products of fermentation; through increasedutilisation of lactic acid; or through the conversion of lactic acid tovolatile fatty acids which can be absorbed from the gut, therebyreducing acidity in the gut.

The effects of immunisation and antibiotic use can be further enhancedby their use in combination with probiotics in the form of bacteriaselected for favourable fermentation characteristics. For example,Megasphera elsdenii and certain strains of Selenomonas ruminantium thatcan ferment sugars or starch without accumulation of lactic acid may beused in a probiotic composition. Other probiotic bacteria specificallyselected for reducing lactic build up during starch fermentation includeLu-12 SU109 and Sulll (Aihua Liu, 1999 see p117). Strains of Selenomonasruminantium vary significantly in their ability to ferment starch and intheir ability to utilise lactic acid. While some strains have beenisolated for their ability to utilise lactic acid, the strains (SRR1 andSRR3) disclosed in the present invention are aggressive producers oflactic acid. This combination of immunisation/antibiotic treatment withthe use of probiotics allows the control of lactic acid producingbacteria and thus allowing the better establishment of favourable starchutilising organisms. The effect of this combination of treatments issynergistic and not merely additive.

Typically, preferred probiotic preparations include bacteria whichferment starch and sugars to end products other than lactic acid, (ievolatile fatty acids), and bacteria which convert lactic acid tovolatile fatty acids. More typically, microorganisms such as Megaspheraelsdentiii and certain strains of Selenomonas ruminantium can fermentsugars or starch without accumulation of lactic acid and these strainscan be used to reduce lactic acid accumulation.

More typically, the probiotic preparations may include bacteria thatbelong to the genera: Succinomonas, Butyrivibrio, Bacteroides andSuccinivibrio. These bacteria can be used individually or incombinations. More typically, the probiotic preparations may includeanaerobic bacteria. Even more typically, the probiotic preparations mayinclude bacteria selected from the group consisting of: Megasphera,Veillenolla, Selenomonas, Propionibacterium, Anaerovibrio andPeptococcus. These bacteria can be used individually or in combinations.Still more typically, preferred probiotic preparations include yeast andmycelial preparations capable of utilising lactic acid, and convertinglactic acid to volatile fatty acids and other end products. Yet stillmore typically, the probiotic preparations may include yeast andmycelial preparations such as Yea-Sacc.

Typically, at least any two of the above sample microorganisms of theprobiotic preparation may be used in combination in the probioticpreparation.

Typically, the above probiotics may be used in conjunction with thevaccine in accordance with the first or seventeenth embodiments of theinvention.

Typically, a combination of immunisation/active agent treatment, allowsthe control harmful lactic acid producing bacteria and thus allowing thebetter establishment of favourable starch utilising organisms. Theeffect of this combination of treatment is synergistic and not merelyadditive.

Typically, lactic acidosis is associated with a wide range of disorders,including: immune disorders, including diabetes, dermatitis, arthritis,rheumatoid arthritis, multiple sclerosis, amyotrophic lateral sclerosis,chronic fatigue syndrome, myasthenia gravis, inflammatory bowel disease,coeliac disease, irritable bowl syndrome, crohn's disease, effects onthe pancreas, kidneys, thryoid and other organs of the endocrine system,and immune conditions associated with localised inflammation of sectionsof the gut; homeostasis disorders, including mineral and electrolyteimbalances, such as osteoporosis; impaired reproductive performance;predisposition to ulceration of the gastrointestinal tract; respiratorytract disorders, including asthma; attention deficit disorder, autism,atopy, hypertension; infected gums and dental caries; viral infections,including herpes; predisposition to infection by bacteria, viruses andmycoplasma fungi or protozoa; exacerbation of heat stress, and impairedhair, milk production and wool growth.

3. Nucleic acid molecules and antibodies

According to a twelfth embodiment of the invention, there is provided anisolated nucleic acid molecule comprising a polynucleotide sequencecapable of selectively hybridising to at least a portion of the nucleicacid of at least one of the isolated microorganisms selected from thegroup consisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates.

Typically, the microorganism is selected from the group consisting of:Streptococcus equinus, Clostridium-like vitulinus, Selenomonasruminantium, Prevotella-like species, Bacteroides-like species,Enterococcus-like species, Streptococcus bovis SbR1 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08. Evenmore typically, the microorganism is selected from the group consistingof: Streptococcus bovis (SbR1), Streptococcus equinus (SER1);Streptococcus equinus (SER2); Selenomonas ruminantium (SRR1);Selenomonas ruminantium (SRR3); Clostridium-like vitulinus (LVR3);Clostridium-like vitulinus (LVR4), Prevotella-like isolates LAB01 andLAB03, Bacteroides-like isolates LAB07, Enterococcus-like isolate LAB05,Streptococcus bovis SbR1, non-dextran slime producing Streptococcusisolate LAB04 and non-slime producing lactic acid bacterial isolatesLAB02, LAB06 and LAB08.

Yet still more typically, the microorganism is selected from the groupconsisting of: Streptococcus bovis (SbR1) (NM99/04455), Streptococcusequinus (SER1) (NM999/04456); Streptococcus equinus (SER2) (NM99/04457);Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonas ruminantium(SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3) (NM99/4461);Clostridium-like vitulinus (LVR4) (NM99/04462), Prevotella-like isolatesLAB01 (NM00/12630) and LAB03 (NM00/12632), Bacteroides-like isolatesLAB07 (NM00/12636), Enterococcus-like isolates LAB05 (NM00/12634),Streptococcus bovis (SbR1), non-dextran slime producing Streptococcusisolate LAB04 (NM00/12633) and non-slime producing lactic acid bacterialisolates LAB02 (NM00/12631), LAB06 (NM00/12635) and LAB08 (NM00/12637).

According to a thirteenth embodiment of the invention, there is providedan isolated nucleic acid molecule comprising a polynucleotide sequenceselected from the group consisting of: SEQ ID Nos: 1–7.

Typically, the nucleic acid molecule corresponds to a DNA or RNAmolecule.

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thetwelfth or thirteenth embodiments of the invention, wherein saidanalogue encodes a polypeptide having a biological activity which isfunctionally the same as the polypeptide(s) encoded by thepolynucleotide sequence defined in accordance with the twelfth orthirteenth embodiments of the invention, wherein said polynucleotidesequence can be located and isolated using standard techniques inmolecular biology, without undue trial and experimentation.

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thetwelfth or thirteenth embodiments of the invention, which has at least45% homology to the polynucleotide sequences so defined. More typically,the analogue of the polynucleotide sequences has at least 55% homology,still more typically the analogue has at least 60% homology, even moretypically, the analogue has at least 75% homology, still more typically,the analogue has at least 85% homology, and yet still more typically,the analogue has at least 90% homology, and yet even still moretypically, the analogue has at least 95–99% homology to thepolynucleotide sequences so defined.

The degree of homology between two nucleic acid sequences may bedetermined by means of computer programs known in the art such as GAPprovided in the GCG program package (Program Manual for the WisconsinPackage, Version 8, August 1996, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48, 443–453). Using GAP with thefollowing settings for DNA sequence comparison: GAP creation penalty of5.0 and GAP extension penalty of 0.3.

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thetwelfth or thirteenth embodiments of the invention, wherein saidanalogue is capable of hybridising to the polynucleotide sequencesdefined in accordance with the twelfth or thirteenth embodiments of theinvention under conditions of low stringency. More typically, lowstringency hybridisation conditions correspond to hybridisationperformed in conditions of low temperature and/or high salt. Even moretypically, low stringency hybridisation conditions correspond tohybridisation performed at 50° C. in 6×SSC.

For example, suitable experimental conditions for determining whether agiven nucleic acid molecule, hybridises to a specified nucleic acid mayinvolve following the following hybridisation routine: presoaking of afilter containing a relevant sample of the nucleic acid to be examinedin 5× SSC for 10 min, and prehybridisation of the filter in a solutionof 5× SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml of denaturedsonicated salmon sperm DNA, followed by hybridisation in the samesolution containing a concentration of 10 ng/ml of a ³²P-dCTP-labeledprobe for 12 hours at approximately 45° C., in accordance with thehybridisation methods as described in Sambrook et al. (1989; MolecularCloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, N.Y.).

The filter is then washed twice for 30 minutes in 2× SSC, 0.5% SDS atleast 55° C. (low stringency), at least 60° C. (medium stringency), atleast 65° C. (medium/high stringency), at least 70° C. (highstringency), or at least 75° C. (very high stringency). Hybridisationmay be detected by exposure of the filter to an X-ray film.

Further, there are many conditions and factors, well known to thoseskilled in the art, which may be used to alter the stringency ofhybridisation. For instance, alterations to features such as: the lengthand nature (DNA, RNA, base composition) of the nucleic acid to behybridised to a specified nucleic acid; concentration of salts and othercomponents, such as the presence or absence of formamide, dextransulfate, polyethylene glycol etc; and altering the temperature of thehybridisation and/or washing steps, all influence the dynamics andstringency of nucleic acid hybridisation.

Further, it is also possible to theoretically predict whether or not twogiven nucleic acid sequences will hybridise under certain specifiedconditions. Accordingly, as an alternative to the empirical methoddescribed above, the determination as to whether an analogous nucleicacid sequence will hybridise to the nucleic acid molecule in accordancewith the twelfth or thirteenth embodiments of the invention, can bebased on a theoretical calculation of the T_(m) (melting temperature) atwhich heterologous nucleic acid sequences with known sequences willhybridise under specified conditions, such as salt concentration andtemperature.

In determining the melting temperature for heterologous nucleic acidsequences (T_(m(hetero))) it is necessary first to determine the meltingtemperature (T_(m(homo))) for homologous nucleic acid sequence. Themelting temperature (T_(m(homo))) between two fully complementarynucleic acid strands (homoduplex formation) may be determined inaccordance with the following formula, as outlined in Current Protocolsin Molecular Biology, John Wiley & Sons, 1995, as:T _(m(homo))=81.5° C.+16.6(log M)+0.41 (%GC)−0.61 (% form)−500 mL

-   -   M=denotes the molarity of monovalent cations,    -   %GC=% guanine (G) and cytosine (C) of total number of bases in        the sequence,    -   % form=% formamide in the hybridisation buffer, and    -   L=the length of the nucleic acid sequence.

T_(m) determined by the above formula is the T_(m) of a homoduplexformation (T_(m(homo))) between two fully complementary nucleic acidsequences. In order to adapt the T_(m) value to that of two heterologousnucleic acid sequences, it is assumed that a 1% difference in nucleotidesequence between two heterologous sequences equals a 1° C. decrease inT_(m). Therefore, the T_(m(hetero)) for the heteroduplex formation isobtained through subtracting the homology % difference between theanalogous sequence in question and the nucleotide probe described abovefrom the T_(m(homo)).

Typically the nucleic acid molecule in accordance with the twelfth orthirteenth embodiments of the invention also includes within its scope anucleic acid molecule which is an oligonucleotide fragment of thesepolynucleotide sequences.

Typically, the oligonucleotide fragment is between about 10 to about 100nucleotides in length. More particularly, the oligonucleotide fragmentis a between about 10 to about 75 nucleotides in length. Even moretypically, the oligonucleotide fragment is between about 15 to about 50nucleotides in length. Even more typically still, the oligonucleotidefragment is between about 15 to about 30 nucleotides in length. Yetstill more typically, the oligonucleotide fragment is between about 5 toabout 25 nucleotides in length.

According to a fourteenth embodiment of the invention, there is provideda vector comprising the nucleic acid molecule in accordance with thetwelfth or thirteenth embodiments of the invention.

Typically, the vector is a shuttle or expression vector. More typically,the vector is selected from the group consisting of: viral, plasmid,bacteriophage, phagemid, cosmid, bacterial artificial chromosome, andyeast artificial chromsome. More typically, the vector is a plasmid andmay be selected from the group consisting of: pBR322, M13mp18, pUC18 andpUC19. Even more typically, the vector is a bacteriophage and may beselected from λgt10 and λgt11 or phage display vectors.

According to a fifteenth embodiment of the invention, there is provideda host cell transformed with the vector in accordance with thefourteenth embodiment of the invention.

Typically, the host cells are procaryotic or eucaryotic in nature. Moretypically, the procaryotic host cells include bacteria, and examples ofsuch bacteria include: E. coli, Bacillus, Streptomyces, Pseudomonoas,Salmonella, and Serraria.

More typically, the eucaryotic host cells may be selected from the groupconsisting of: yeast, fungal, plant, insect cells and mammalian cells,either in vivo or in tissue culture. Examples of mammalian cellsinclude: CHO cell lines, COS cell lines, HeLa cells, L cells, murine 3T3cells, c6 glioma cells and myeloma cell lines.

According to a sixteenth embodiment of the invention, there is providedan antibody raised against at least one lactic acid producingmicroorganism, wherein said microorganism is selected from the groupconsisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates.

Typically, the microorganism is selected from the group consisting of:Streptococcus equinus, Clostridium-like vitulinus, Selenomonasruminantium, Pravotella-like species, Bacteroides-like species,Enterococcus-like species, Streptococcus bovis SbR1 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08. Evenmore typically, the microorganism is selected from the group consistingof: Streptococcus bovis (SbR1), Streptococcus equinus (SER1);Streptococcus equinus (SER2); Selenomonas ruminantium (SRR1);Selenomonas ruminantium (SRR3); Clostridium-like vitulinus (LVR3);Clostridium-like vitulinus (LVR4), Prevotella-like isolates LAB01 andLAB03, Bacteroides-like isolates LAB07, Enterococcus-like isolate LAB05,Streptococcus bovis SbR1, non-dextran slime producing Streptococcusisolate LAB04 and non-slime producing lactic acid bacterial isolatesLAB02 LAB 06 and LAB08.

Yet still more typically, the microorganism(s) is selected from thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456): Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462),Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like LAB07 (NM00/12636), Enterococcus-like isolate LAB05(NM00/12634), Streptococcus bovis (SbR1), non-dextran slime producingStreptococcus isolate LAB04 (NM00/12633) and non-slime producing lacticacid bacterial isolates LAB02 (NM00/12631), LAB06 (NM00/12635) and LAB08(NM00/12637).

Typically, the antibody in accordance with the sixteenth embodiment ofthe invention is raised against at least one of the following:

-   (a) at least one fermentative lactic acid producing microorganism as    defined in the sixteenth embodiment of the invention;-   (b) intact cells of least one fermentative lactic acid producing    microorganism as defined in the sixteenth embodiment of the    invention;-   (c) cell lysate from at least one fermentative lactic acid producing    microorganism as defined in the sixteen embodiment of the invention;-   (d) crude antigen mixture or purified antigen or antigens from at    least one fermentative lactic acid producing microorganism as    defined in the sixteenth embodiment of the invention;-   (e) outer membrane and associated proteins of at least one    fermentative lactic acid producing microorganism as defined in the    sixteenth embodiment of the invention.

Typically, the antibodies in accordance with the sixteenth embodimentmay be present in a composition further comprising antibodies raisedagainst at least one of the following:

-   (f) at least one fermentative lactic acid producing strain selected    from Streptococcus bovis or lactobacilli;-   (g) intact cells of at least one fermentative lactic acid producing    strain selected from Streptococcus bovis or lactobacilli;-   (h) cell lysate from at least one fermentative lactic acid producing    strain selected from Streptococcus bovis or lactobacilli;-   (i) crude antigen mixture or purified antigen or antigens from at    least one fermentative lactic acid producing strain selected from    Streptococcus bovis or lactobacilli;-   (j) outer membrane and associated proteins of at least one    fermentative lactic acid producing strain selected from    Streptococcus bovis or lactobacilli.

More typically, the antibodies in accordance with the sixteenthembodiment may be present in a composition further comprising antibodiesraised against at least one of the following:

-   (k) Streptococcus bovis (strain Sb-5) deposited with the Australian    Government N94/8255;-   (l) intact cells of Streptococcus bovis (strain Sb-5) deposited with    the Australian Government Analytical Laboratories (AGAL) on 8 Mar.    1994, and given accession number N94/8255;-   (m) cell lysate from Streptococcus bovis (strain Sb-5) deposited    with the Australian Government Analytical Laboratories (AGAL) on 8    Mar. 1994, and given accession number N94/8255;-   (n) crude antigen mixture or purified antigen or antigen from    Streptococcus bovis (strain Sb-5) deposited with the Australian    Government Analytical Laboratories (AGAL) on 8 Mar. 1994, and given    accession number N94/8255;-   (o) outer membrane and associated proteins of Streptococcus bovis    (strain Sb-5) deposited with the Australian Government Analytical    Laboratories (AGAL) on 8 Mar. 1994, and given accession number    N94/8255.

Typically, the antibodies in accordance with the sixteenth embodiment ofthe invention can be comprised of a polyclonal mixture, or may bemonoclonal in nature. Further, antibodies can be entire immunoglobulinsderived from natural sources, or from recombinant sources. Theantibodies of the present invention may exist in a variety of forms,including for example as a whole antibody, or as an antibody fragment,or other immunologically active fragment thereof, such ascomplementarity determining regions.

The antibody (or fragment thereof) in accordance with the sixteenthembodiment of the present invention has binding affinity to amicroorganisms capable of producing acid in the gut of vertebrates invertebrates. Preferably, the antibody (or fragment thereof) has bindingaffinity or avidity greater than about 10⁵ M⁻¹, more preferably greaterthan about 10⁶ M⁻¹, more preferably still greater than about 10⁷ M⁻¹ andmost preferably greater than about 10⁸ M⁻¹.

The techniques for generating and reviewing binding affinity arereviewed in Scatchard (1949). Annals of the New York Academy ofSciences, 51, 660–672, and Munson (1983). Methods in Enzymology 92,543–577, the contents of each of which are incorporated herein byreference.

According to a seventeenth embodiment of the invention, there isprovided a vaccine comprising at least one of the antibodies inaccordance with the sixteenth embodiment of the invention together witha pharmaceutically acceptable carrier, adjuvant and/or diluent.

4. Diagnostic of Acidosis

According to an eighteenth embodiment of the invention, there isprovided a diagnostic kit for the detection of microorganisms having arole in lactic acidosis in a vertebrate, said kit comprising at leastone of the antibodies in accordance with the sixteenth embodiment of theinvention, together with a diagnostically acceptable carrier and/ordiluent.

Typically, the diagnostic kit may also contain antibodies capable ofdetecting at least one lactic acid producing strain selected from:Clostridium-like species, Prevotella-like species, Bacteroides-likespecies, Enterococcus-like species, Selenomonas species, non-dextranslime producing Streptococcus species and non-slime producing lacticacid bacterial isolates.

Typically, the kits will also include analytical methods to measure theextent of tactic acid production, specifically lactic acid in order todetermine functional aspects of the lactic acid producing bacteria. Moretypically, the kit also contains reagents for measuring acidity andcapacity to produce lactic acid as well as the detection of themicroorganisms responsible for the lactic acid production. The kit maycontain reagents and equipment to measure pH of digesta or fascalmaterial and fermentation tubes for measuring the potential lactic acidproduction with added carbohydrate. Both pH and lactic acid aretypically detected using colour or other visual changes,spectrophotometric methods or through instruments such as pH metes.

More typically, the diagnostic kit may also contain antibodies capableof detecting microorganisms selected from the group consisting of:Clostridium-like species, Prevaotella-like species, Bacteroides-likespecies, Enterococcus-like species, Selenomonas species, non-dextranslime producing Streptococcus species and non-slime producing lacticacid bacterial isolates.

Even more typically, the diagnostic kit may also contain antibodiescapable of detecting miroorganisms selected from the group consistingof: Streptococcus equinus, Clostridium-like vitulinus, Selenomonasruminantium, Prevotella-like species, Bacteroides-like species,Enterococcus-like species, Streptococcus bovis SbR1 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08. Evenmore typically, the microorganism is selected from the group consistingof: Streptococcus bovis (SbR1), Streptococcus equinus (SER1);Streptococcus equinus (SER2); Selenomonas ruminantium (SRR1);Selenomonas ruminantium (SRR3); Clostridium-like vitulinus (LVR3);Clostridium-like vitulinus (LVR4), Prevotella-like isolates LAB01 andLAB03, Bacteroides-like isolates LAB07, Enterococcus-like isolate LAB05,Streptococcus bovis SbR1, non-dextran slime producing Streptococcusisolate LAB04 and non-slime producing lactic acid bacterial isolatesLAB02, LAB06 and LAB08.

Yet still more typically, the microorganism(s) are selected from thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462);Prevotella-like LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like isolate LAB07 (NM00/12636); Enterococcus-like isolateLAB05 (NM00/12634), Streptococcus bovis (SbR1), non-dextran slimeproducing Streptococcus isolate LAB04 (NM00/12633) and non-slimeproducing lactic acid bacterial isolates LAB02 (NM00/12631), LAB06(NM00/12635) and LAB08 (NM00/12637), together with Streptococcus bovis(strain Sb-5) deposited with the Australian Government AnalyticalLaboratories (AGAL) on 8 Mar. 1994, and given accession number N94/8255.

Typically, the kit may comprise the following containers:

(a) a first container containing at least the antibody (or fragmentthereof) in accordance with the sixteenth embodiment of the invention,and;

(b) a second container containing a conjugate comprising a bindingpartner of the antibody (or fragment thereof), together with adetectable label.

More typically, the first container containing at least the antibody (orfragment thereof) in accordance with the sixteenth embodiment of theinvention, may further comprise antibodies selected from the groupconsisting of: antibodies capable of detecting at least one lactic acidproducing strain selected from Streptococcus bovis or lactobilli andantibodies capable of detecting Streptococcus bovis (strain Sb-5)deposited with the Australian Government Analytical Laboratories (AGAL)on 8 Mar. 1994, and given accession number N94/8255.

More typically, the kit may further comprise one or more othercontainers, containing other components, such as wash reagents, andother reagents capable of detecting the presence or bound antibodies.Even more typically, the detection reagents may include: labelled(secondary) antibodies, or whether the antibody (or fragment thereof) inaccordance with the sixteenth embodiment of the invention is itselflabelled, the compartment may comprise antibody binding reagents capableof reacting with the labelled antibody (or fragments thereof) of thepresent invention.

According to a nineteenth embodiment of the invention, there is provideda method for screening for the presence of microorganism having a rolein acidosis in a vertebrate, said method comprising:

-   containing a sample from the gut of a vertebrate with the antibody    (or fragment thereof) in accordance with the sixteenth embodiment of    the invention; and-   (b) detecting the presence of the antibody (or fragment thereof)    bound to microorganisms having a role in acidosis.

Typically, the antibody used in the method in accordance with thenineteenth embodiment of the invention corresponds to an antibody mix,comprising antibody or fragment thereof in accordance with the sixteenthembodiment of the invention, together with an antibody(s) selected fromthe group consisting of: antibodies capable of detecting at least onelactic acid producing strain selected from Streptococcus bovis orlactobacilli and antibodies capable of detecting Streptococcus bovis(strain Sb-5) deposited with the Australian Government AnalyticalLaboratories (AGAL) on 8 Mar. 1994, and given accession number N94/8255.

According to a twentieth embodiment of the invention, there is provideda method for screening for the presence of microorganisms having a rolein acidosis in a vertebrate, said method comprising contacting a nucleicacid sample from a microorganism with a nucleic acid probe, wherein themicroorganism is selected from the group consisting of: Clostridium-likespecies, Prevotella-like species, Bacteroides-like species,Enterococcus-like species, Selenomonas species, non-dextran slimeproducing Streptococcus species and non-slime producing lactic acidbacterial isolates, isolated from the gut of a vertebrate, and

(b) detecting hybridisation between the nucleic acid sample and thepolynucleotide sequence.

Typically, the microorganism is selected from the group consisting of:microorganisms selected from the group consisting of: Streptococcusequinus, Clostridium-like vitulinus, Selenomonas ruminantium,Prevotella-like species, Bacteroides-like species, Enterococcus-likespecies, Streptococcus bovis SbR1 and non-slime producing lactic acidbacterial isolates LAB02, LAB06 and LAB08. Even more typically, themicroorganism is selected from the group consisting of: Streptococcusbovis (SbR1), Streptococcus equinus (SER1); Streptococcus equinus(SER2); Selenomonas ruminantium (SRR1); Selenomonas ruminantium (SRR3);Clostridium-like vitulinus (LVR3); Clostridium-like vitulinus (LVR4);Prevotella-like isolates LAB01 and LAB03, Bacteroides-like isolatesLAB07, Enterococcus-like isolate LAB05, Streptococcus bovis SbR1,non-dextran slime producing Streptococcus isolate LAB04 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08.

Yet still more typically, the microorganism(s) is selected form thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462),Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like isolate LAB07 (NM00/12636), Enterococcus-like isolateLAB05 (NM00/12634), Streptococcus bovis (SbR1), non-dextran slimeproducing Streptococcus isolate LAB04 (NM00/12 631), LAB06 (NM00/12635)and LAB08 (NM00/12637).

Typically, hybridisation as compared to non-hybridisation is indicativeof the presence of microorganisms having a role in acidosis.

Typically, the nucleic acid probe corresponds to a portion of thepolynucleotide sequence in accordance with the twelfth or thirteenthembodiments of the invention which is capable of selectively hybridisingto nucleic acid from a sample of gut microorganisms as defined in thefirst embodiment of the invention. More typically, the nucleic acidprobe corresponds to a probe mix, comprising a portion of thepolynucleotide sequence in accordance with the twelfth or thirteenthembodiments of the invention which is capable of selectively hybridisingto nucleic acid from a sample, together with an isolated nucleic acidmolecule comprising a polynucleotide sequence capable of selectivelyhybridising to the nucleic acid (or portion thereof) of at least onelactic acid producing strain selected from Streptococcus bovis orlactobacilli, or a polynucleotide sequence capable of selectivelyhybridising to the nucleic acid (or portion thereof) of themicroorganism strain Streptococcus bovis (strain Sb-5) deposited withthe Australian Government Analytical Laboratories (AGAL) on 8 Mar. 1994,and given accession number N94/8255.

Typically, hybridisation may occur and be detected through techniquesthat are routine and standard amongst those skilled in the art, andinclude southern and northern hybridisation, polymerase chain reaction(PCR) and ligase chain reaction (LCR) amplification.

Various low or high stringency hybridisation levels may be used,depending on the specificity and selectivity desired.

Typically, the microorganisms detected in accordance with the nineteenthor twentieth embodiment of the invention may also include lactic acidproducing strain selected from Streptococcus bovis or lactobacilli. Moretypically, the microorganisms detected in accordance with the nineteenthor twentieth embodiment of the invention nay include Streptococcus bovis(strain Sb-5) deposited with the Australian Government AnalyticalLaboratories (AGAL) on 8 Mar. 1994, and given accession number N94/8255.

According to a twenty-five embodiment of the invention, there isprovided a method for screening for potential therapeutic agents for thetreatment of lactic acidosis in a vertebrate, said method comprising

-   (a) contacting the potential therapeutic agent with a microorganism    selected from the group consisting of: Clostridium-like species,    Prevotella-like species, Bacteroides-like species, Enterococcus-like    species, Selenomonas species, non-dextran slime producing    Streptococcus species and non-slime producing lactic acid bacterial    isolates, and-   (b) detecting an effect of the potential therapeutic agent on said    microorganism.

Typically, the microorganism is selected from the group consisting of:microorganism selected from the group consisting of: Streptococcusequinus, Clostridium-like vitulinus, Selenomonas ruminantium,Prevotella-like species, Bacteroides-like species, Enterococcus-likespecies, Streptococcus bovis SbR1 and non-slime producing lactic acidbacterial isolates LAB02, LAB06 and LAB08. Even more typically, themicroorganism is selected from the group consisting of: Streptococcusbovis (SbR1), Streptococcus equinus (SER1); Streptococcus equinus(SER2); Selenomonas ruminantium (SRR1); Selenomonas ruminantium (SSR3);Clostridium-like vitulinus (LVR3); Clostridium-like vitulinus (LVR4),Prevotella-like isolates LAB01 and LAB03, Bacteroides-like isolatesLAB07, Enterococcus-like isolate LAB05, Streptococcus bovis SbR1,non-dextran slime producing Streptococcus isolate LAB04 and non-slimeproducing lactic acid bacterial isolates LAB02, LAB06 and LAB08.

Yet still more typically, the microorganism(s) is selected from thegroup consisting of: Streptococcus bovis (SbR1) (NM99/04455),Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1), (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462),Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632),Bacteroides-like isolate LAB07 (NM00/12636), Enterococcus-like isolateLAB05 (NM00/12634), Streptococcus bovis (SbR1), non-dextran slimeproducing Streptococcus isolates LAB04 (NM00/12633) and non-slimeproducing lactic acid bacterial isolates LAB02 (NM00/12631), LAB06(NM00/12635) and LAB08 (NM00/12637).

Typically, the screening method determines whether the potentialtherapeutic agent has a role in a lactic acid production pathway in atleast one of the microorganisms of the invention. For example, thescreening method in accordance with the twenty-first embodiment of theinvention may identify a therapeutic agent which affects a critical stepin the lactic acid biosynthetic pathway, such as blocking the conversionof pyruvate to lactate by inactivating the enzyme dehydrogenase, orblocking the conversion of malate to lactate by inactivating malo-lacticenzyme.

According to a twenty-second embodiment of the invention, there isprovided an isolated culture of at least one microorganism selected fromthe group consisting of: Streptococcus bovis (SbR1) (NM99/04455);Streptococcus equinus (SER1) (NM99/04456); Streptococcus equinus (SER2)(NM99/04457); Selenomonas ruminantium (SRR1) (NM99/04458); Selenomonasruminantium (SRR3) (NM99/04460); Clostridium-like vitulinus (LVR3)(NM99/04461); Clostridium-like vitulinus (LVR4) (NM99/04462);Prevotella-like isolates LAB01 (NM00/12630) and LAB03 (NM00/12632);Bacteroides-like isolate LAB07 (NM0-/12636); Enterococcus-like isolatesLAB05 (NM00/12634), Streptococcus bovis (SbR1); non-dextran slime acidbacterial isolates LAB02 (NM00/12631), LAB06 (NM00/12635) and LAB08(NM00/123637).

The term “antibody” means an immunoglobulin molecule able to bind to aspecific epitope on an antigen. Antibodies can be comprised of apolyclonal mixture, or may be monoclonal in nature. Further, antibodiescan be entire immunoglobulins derived from natural sources, or fromrecombinant sources. The antibodies of the present invention may existin a variety of forms, including for example as a whole antibody, or asan antibody fragment, or other immunologically active fragment thereof,such as complementarity determining regions. Similarly, the antibody mayexist as an antibody fragment having functional antigen-binding domains,that is, heavy and light chain variable domains. Also, the antibodyfragment may exist in a form selected from the group consisting of: Fv,F_(ab), F(ab)₂, scFv (single chain Fv), dAb (single domain antibody),bi-specific antibodies, diabodies and triabodies.

The term “isolated” means that the material in question has been removedfrom its host and associated impurities reduced or eliminated.Essentially, it means an object species is the predominant speciespresent (ie., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably a substantiallypurified fraction is a composition wherein the object species comprisesat least about 30 percent (on a molar basis) of all macromolecularspecies present. Generally, a substantially pure composition willcomprise more than about 80 to 90 percent of all macromolecular speciespresent in the composition. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

As used herein the term “polypeptide” means a polymer made up of aminoacids linked together by peptide bonds.

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Further, variationsof the word “comprising”, such as “comprise” and “comprises” havecorrespondingly varied meanings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes a phylogenetic tree illustrating the phylogeneticrelationship of the bacterial species and strains of the presentinvention.

BEST MODE OF PERFORMING THE INVENTION

1. Isolation of Microorganisms involved in acidosis

The following method provides a means of isolating microorganismsinvolved in acidosis. Fluid samples are taken from caecal, colonic,rectal or faecal material from animals or humans consuming a dietcontaining more than half of the dry matter as sugars, oligosaccharidesor starch. This material (one part) may then be mixed well withdistilled water (9 parts), prior to straining through 4 layers of cheesecloth and serially diluting in ten-fold steps using anaerobic dilutionsolution (ADS) (Caldwell and Bryant, 1966) to a final dilution of 10⁻⁸.

The material so diluted (10⁻⁶, 10⁻⁷ and 10⁻⁸) is then used to inoculatemedia roll tubes prepared with modified semi-selective MRS-Agar medium,Oxoid, England, (de Man et al., 1960), together with adjustment of thepH of the medium to 5.5 Three replicate tubes are used for each dilutionand they are incubated at 39° C. for three days.

The colonies so prepared are then carefully studied under a low power(×4) microscope to identify the most common colonies based on physicalappearance and growth characteristics. These colonies are thenenumerated at the three dilutions (10⁻⁶, 10⁻⁷ and 10⁻⁸) to confirm aconsistent representation. At this stage, samples are taken of at leastfive colonies that have been counted as being of the same most commoncharacteristics to confirm similarity, and are examined under high powermagnification (>40×) using gram straining to determine that the cellsare similar.

Once this is done, viable colonies representing the dominant copy typeare picked and used to inoculate a broth of basal medium 10 containingglucose (0.5%). This process of inoculation is repeated into MRS rolltubes and again examined for uniformity among colonies. At this stage,at least three examples of the most common colonies are examined and ifthese appear identical, representative colonies are picked and used toinoculate a broth of BM10. The process of roll tube and broth culturesis repeated until it is clear that a purified isolate has been obtained.In some cases, it is possible that two or more bacteria are very closelyassociated and in this case a crude isolate is maintained as theantigenic unit.

In the case of Streptococcus isolates, dextran (slime) characteristicsare examined by centrifugation at 17,000 g for15 minutes. The absence ofa bacterial “pellet” following centrifugation indicates a dextran typeslime. One of the aims of the present invention was the selection for S.bovis bacterial that did not produce dextran slime.

Finally, characteristics of the isolate can then be determined bymeasuring the range of substrate utilisation and rate of lactic acidproduction.

2. Vaccine/Pharmaceutical Composition and Methods for Control ofAcidosis

In a process of preparing a vertebrate vaccine of the invention, atypical protocol includes: washing the microbial growth free of nutrientmedium, killing, harvesting and suspension of the dead cells of themicroorganisms in a pharmaceutically/veterinarily acceptable carrier,diluent, excipient and/or adjuvant.

An alternative typical protocol includes: washing the microbial growthfree of nutrient medium, rupturing to form outer membrane and associatedproteins, separating whole cells from outer membranes and associatedproteins, suspension of the outer membrane and associated proteins in apharmaceutically/vertinarily acceptable carrier, diluent, excipientand/or adjuvant.

In delivery systems utilising the parenteral route it is preferred thatdead cells of the microorganisms and/or outer membrane and associatedproteins, are suitably washed, harvested and resuspended in apharmaceutically/veterinarily acceptable carrier, diluent and/oradjuvant suitable for injection, utilising methods of administration asare well known in the art.

In the administration of therapeutic formulations in accordance with thepresent invention and herein disclosed, there are preferred non-toxicpharmaceutical carriers, diluents, excipients and/or adjuvants. Foradministration of the above formulations the microorganism or fragmentor fragments thereof of the present invention are admixed with thesenon-toxic carriers, diluents, excipients and/or adjuvants and may be inthe form of capsules, aqueous or oily suspensions, emulsions, micellesor injectable solutions.

Examples of pharmaceutically and veterinarily acceptable carriers ordiluents are demineralised or distilled water; saline solution;vegetable based oils such as peanut oil, safflower oil, olive oil,cottonseed oil, maize oil, sesame oils such as peanut oil, saffloweroil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil orcoconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane;volatile silicones; mineral oils such as liquid paraffin, soft paraffinor squalane; cellulose derivatives such as methyl cellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin;fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrridone; agar; crrageenan; gum tragcanth or gumacacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the compositions.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides, cytokines and buffering agents.

In general to induce the production of antibodies to the vaccines of theinvention, they can be oleogenous or aqueous suspensions formulated inaccordance with known methods in the art using suitable dispersing,suspension and/or wetting agents. Examples of suitable dispersing,suspension and wetting agents include Freund's complete/incompleteadjuvant. Montenide Marcol adjuvant and phosphate buffered saline, andmannan.

it will be appreciated that the examples referred to above areillustrative only and other suitable carriers, diluents, excipients andadjuvants known in the art may be employed without departing from thespirit of the invention.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include. Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and 1,2propylene glycol.

Further, a vaccine composition containing a recombinant polypeptide asencoded by at least one of the nucleic acid molecules in accordance withthe twelfth or thirteenth embodiment of the invention, may be preparedfor use by standard methods, well known to those of ordinary skill inthe art.

In one embodiment, the immunogenic polypeptide, glycopeptide or thelike, may be produced in a recombinant system by expression of thepolynucleotide sequence (or a fragment thereof) in accordance with thetwelfth or thirteenth embodiments of the invention, and subsequentlyisolated. For example, microbial cells containing the nucleic acidmolecule of interest may be cultured in large volume bioreactors, thencollected by centrifugation and subsequently ruptured, for instance byhigh-pressure homogenisation. The resulting cell lysate may beresuspended in appropriate diluent such as those described herein, andfiltered to obtain an aqueous suspension of the immunogen. Therecombinant protein can be administered in crude form, for example, bydiluting in a 0.1M phosphate buffer (pH 7.4) to 50–500 μg/mlconcentration, and then passing through a sterile 0.22 micron filter.

Alternatively, a vaccine composition containing the recombinantimmunogenic polypeptide, glycopeptide or the like, may be prepared in amammalian expression system, utilising host cells such as ChineseHamster Ovary (CHO) cells. The recombinant polypeptide, glycopeptide orthe like, (or fragment thereof) may be manufactured using batchfermentation with serum free medium. After fermentation the recombinantpolypeptide, glycopeptide or the like, (or fragment thereof) may bepurified via a multistep procedure incorporating chromatography andviral inactivation/removal steps. For instance, the recombinantpolypeptide, glycopeptide or the like, (or fragment thereof) may befirst separated by Protein A affinity chromatography and then treatedwith solvent/detergent to inactivate any lipid enveloped viruses.Further purification, typically by anion and cation exchangechromatography may be used to remove residual proteins,solvents/detergents and nucleic acids. The purified recombinantpolypeptide glycopeptide or the like, (or fragment thereof) may befurther purified and formulated into 0.9% saline using gel filtrationcolumns. The formulated bulk preparation may then be sterilised andviral filtered and dispensed.

Alternatively, a vaccine composition containing an immunogenicpolypeptide, glycopeptide or the like, of the microorganism of thepresent invention may be prepared by synthesis of a peptide, usingstandard methods known to those in the art, such as by automatedsynthesis on, for instance, an Applied Biosystems model 430A. Forexample, the peptide may comprise selected amino acid regions of the CDRand/or FR of the polypeptide of the invention. The synthetic peptide canbe administered, for example, after diluting in a 0.1M phosphate buffer(pH 7.4) to 50–500 μg/ml concentration, and passing through a sterile0.22 micron filter.

Alternatively, the vaccine may be a DNA based vaccine. In one aspect,the DNA based vaccine may comprise naked DNA comprising a nucleic acidencoding an immunogenic polypeptide of the microorganism of the presentinvention, or a fragment thereof.

In another aspect, the DNA based vaccine may comprise a nucleic acidmolecule encoding an immunogenic polypeptide of the microorganism of thepresent invention, or a fragment thereof, cloned into an expressionvector. Typically, the expression vector is a eucaryotic expressionvector and may include expression control sequences, such as an originof replication, a promoter, an enhancer, and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.

A typical vaccination regime is to deliver the vaccine in multiple dosesgenerally one, two or three equal doses.

The vaccines of the invention are typically formulated foradministration by parenteral route, by inhalation or topically, The termparenteral as used herein includes intravenous, intradermal,intramuscular, subcutaneous, rectal, vaginal or intraperitonealadministration. The subcutaneous and intramuscular forms of parenteraladministration are generally preferred.

A vaccine or pharmaceutical composition of the invention may also beadministered topically, such as externally to the epidermis, to thebuccal cavity and instillation of such an antibody into the ear, eye andnose.

The amount of the vaccine or pharmaceutical composition of the inventionrequired for therapeutic or prophylactic effect will, of course, varywith the vaccine or pharmaceutical composition chosen, the nature andseverity of the condition being treated and the animal undergoingtreatment, and is ultimately at the direction of the physician orveterinarian. A suitable topical dose of a vaccine or pharmaceuticalcomposition of the invention will generally be within the range of about1 to about 100 milligrams per kilogram body weight daily; preferablyabout 0.05 to about 50, more preferably about 0.5 to about 25, even morepreferably about 0.5 to about 10 milligrams per kilogram body weight perday.

The parenteral dosage regimens for employing compounds of the inventionto prophylactically or therapeutically control lactic acidosis willgenerally be in the range of about 0.01 to about 100, preferably about0.01 to about 50, more preferably about 0.05 to about 25, even morepreferably about 0.1 to about 2 milligrams per kilogram body weight perday. Alternatively, dosage rates can be determined in relation tometabolic rate or surface area of the body.

A vaccine or pharmaceutical composition of the invention may also beadministered by inhalation, that is, intranasal and/or inhalationadministration. Appropriate dosage forms for such administration, suchas an aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques. The preferred dosage amount of a compound ofthe invention to be employed is generally within the range of about 0.05to about 100, preferably about 0.05 to about 50, more preferably about0.5 to about 25, even more preferably about 0.5 to about 10 milligramsper kilogram body weight per day.

Typically, the dosage rate for immunisation is between 1×10⁶ and 1×10¹¹bacterial cells per administration.

Typically, the dosage rates are approximately equivalent to between1×10⁸ to 1×10⁹ bacterial cells per kg body weight. More typically, thedosage rates are approximately equivalent to between 1×10⁸ and 5×10⁸bacterial cells per kg body weight. Even more typically, the dosagerates are approximately equivalent to 2.5×10⁸ bacterial cells per kgbody weight.

Typically, the dosage rate for immunisation of small animals, such assheep, is between 1×10⁹ and 5×10¹⁰ bacterial cells per injection. Moretypically, the dosage rate for immunisation of small animals, such assheep, is approximately 5×10⁹ bacterial cells per administration.

Typically, the dosage rate for immunisation of large animals, such ascattle and horses, is between 1×10⁹ and 1×10¹² bacterial cells perinjection. More typically, the dosage rate for immunisation of largeanimals, such as cattle and horses, is approximately 1×10¹⁰ bacterialcells per administration.

Typically, the injection volume for sheep is between 1 mL to 3 mL, and 2to 7 mL for cattle and horses 3 to 5 mL. More typically, the injectionvolume for sheep is between 1 mL to 2 mL, and 1 to 5 mL for cattle andhorses.

In accordance with any one of the third through eleventh embodiments ofthe invention, the administered dose of the antibiotic can vary and willdepend on several factors, such as the condition, age and size of thehuman or animal patient, as well as the nature of the lactic acidproducing bacteria.

Dosages will typically range from between any one of the following: 0.01and 100 mg per kg of bodyweight; 0.01 and 75 mg per kg of bodyweight;0.01 and 50 mg per kg of bodyweight; 0.01 and 25 mg kg of bodyweight;0.01 and 15 mg per kg of bodyweight; 0.01 and 10 mg per kg ofbodyweight; and 0.01 and 5 mg per kg of bodyweight. More typicallydosages will range from between 0.2 and 2.0 mg per kg of bodyweight.More typically dosages will range from between 0.5 and 1.0 mg per kg ofbodyweight. Even more typically dosages will range from between 0.1 and0.5 mg per kg of bodyweight. Yet even more typically, the antibiotic isadministered to the human or animal at a rate of 0.4 mg per kg ofbodyweight.

Typically, the antibiotic is administered at a rate of between 1 and 100mg per kg of dry weight of food. More typically, the antibiotic isadministered at a rate of between 1 and 75 mg per kg of dry weight orfood. Even more typically, the antibiotic is administered at a rate ofbetween 1 and 50 mg per kg of dry weight of food. Yet even moretypically, the antibiotic is administered at a rate of between 5 and 40mg per kg of dry weight of food.

Typically, antibiotic preparations are selected and/or formulated fordelivery to the hind gut and for little or no absorption from thedigestive tract. Formulations include encapsulation and/or coating withmaterials resistant to acid and enzymatic digestion in the stomach andsmall intestine. Formulation can also include chemical treatment toreduce the solubility of the antibiotic.

As above, the administered dose of the enzyme preparation can vary andwill depend on several factors, such as the condition, age and size ofthe human or animal patient, as well as the nature of the carbohydrate.Dosages will typically range from between 0.01 and 50 g/kg food drymatter. Typically, the enzyme is administered at a rate of between 0.1and 3 g per kg of dry weight of food. More typically, the enzyme isadministered at a rate of between 1 g per kg of dry weight of food.

Similarly, the administered dose of the clay preparation can vary andwill depend on several factors, such as the condition, age and size ofthe human or animal patient, as well as the nature of the carbohydrate.Dosages will typically range from between 0.5 and 100 g/kg food drymatter. Typically, the clay is administered at a rate of between 1 and50 g per kg of the dry weight of food. More typically, the clay isadministered at a rate of between 10 and 20 g per kg of dry weight offood.

Typically, the administered dose of the probiotic preparation can varybetween 10⁴ and 10¹² bacteria per kg of body weight. More typically,dose of the probiotic preparation can vary between 10⁴ and 10¹⁰ per kgof body weight. Even more typically, dose of the probiotic preparationcan vary between 10⁴ and 10⁶ per kg of body weight.

Typically, probiotics are formulated in such a way as to deliver viablebacteria and/or other microorganisms to gastrointestinal tracerincluding the hind gut. These formulation techniques include coatingsand encapsulation using materials resistant to gastric and intestinaldigestion.

According to another form of the invention, the active agents can beused together.

According to another aspect of the invention, the formulation of theactive agent ensures that it is administered in a palatable form to theanimal or human and in a form which retains activity and is properlymixed in the appropriate compartment(s) of the gastrointestinal tract.

Generally, the active agent is administered regularly throughout theperiod the animal or human is subjected to a high carbohydrate diet orto sugars or other fermentable compounds which are not efficientlyabsorbed prior to reaching the large intestine, colon and caecum.

More typically, the active agent is administered 1–3 times daily. Evenmore typically, the active agent is administered once daily or can beincluded in human food and animal feeds. They can be fed as powders orsuspended in water, included in pellets as well as being fed inpremixes.

More typically the active agent is mixed with the food, or is added tofeeds which contain starch or sugars which may produce an acidic patternof fermentation in the gastrointestinal tract. The active agent can alsobe added to water included in tablets and the like.

A suitable treatment may include the administration of a single dose ormultiple doses. Usually, the treatment will consist of administering onedose daily of the active agent for a period sufficient to control theaccumulation of acid by fermentation of the carbohydrate in thegastrointestinal tract. Dosing may continue while sources ofcarbohydrate known to cause problems of acidic fermentation in thegastrointestinal tract are included in the diet.

More typically the active agent may be administered in a single doseimmediately before consuming meals containing sources of carbohydratewhich are poorly digested and rapidly fermented.

More typically, the active agent is administered for only day prior toand daily during the consumption of excessive quantities of food stuffscontaining readily fermentable carbohydrates.

Typically, the active agent is administered orally.

3. Antibiotics

Antibodies or immunoglobulins are typically composed of four covalentlybound peptide chains. For example, an IgG antibody has two light chainsand two heavy chains. Each light chain is covalently bound to a heavychain. In turn each heavy chain is covalently linked to the other toform a “Y” configuration, also known as an immunoglobulin conformation.Fragments of these molecules, or even heavy or light chains alone, maybind antigen.

A normal antibody heavy or light chain has an N-terminal (NH₂) variable(V) region, and a C-terminal (COOH) constant (C) region. The heavy chainvariable region is referred to as V_(H) (including, for example, V_(γ)),and the light chain variable region is referred to as V_(L) (includingV_(κ) or V_(λ)). The variable region is the part of the molecule thatbinds to the antibody's cognate antigen, while the Fc region (the secondand third domains of the C region) on the heavy chain determines theantibody's effector function (eg. complement fixation, opsonization).Full-length immunoglobulin or antibody “light chains” are encoded by avariable region gene at the N-terminus and a κ (kappa) or λ (lambda)constant region gene at the COOH-terminus. Full-length immunoglobulin orantibody “heavy chains”, are similarly encoded by a variable region geneand one of the constant region genes, e.g., gamma. Typically, the“V_(L)” will include the portion of the light chain encoded by the V_(L)and J_(L) (J or joining region) gene segments and the “V_(H)” willinclude the portion of the heavy chain encoded by the V_(H), and D_(H)(D or diversity region) and J_(H) gene segments.

An immunoglobulin, light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled complementarity-determining regions or CDRs. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three dimensionalspace. The CDRs are primarily referred to as CDR1, CDR2, and CDR3,numbered sequentially starting from the N-terminus.

The two types of light chains, κ (kappa) and λ (lambda), are referred toas isotopes. Isotypic determinants typically reside in the constantregion of the light chain, also referred to as the C_(L) in general, andC_(κ) or C_(λ) in particular. Likewise, the constant region of the heavychain molecule, also known as C_(H), determines the isotype of theantibody. Antibodies are referred to as IgM, IgD, IgG, IgA, and IgEdepending on the heavy chain isotype. The isotopes are encoded in the λ(mu), δ (delta), γ (gamma), α (alpha), and ε (epsilon) segments of theheavy chain constant region, respectively.

The heavy chain isotopes determine different effector functions of theantibody, such as opsonisation or complement fixation. In addition, theheavy chain isotype determines the secreted form of the antibody.Secreted IgG, IgD, and IgE isotypes are typically found in single unitor monomeric form. Secreted IgM isotype is found in pentameric form;secreted IgA can be found in both monomeric and dimeric form.

In a related aspect, the invention features a monoclonal antibody, or anFab, (Fab)₂, scFv (single chain Fv), dAb (single domain antibody),bi-specific antibodies, diabodies and triabodies, or otherimmunologically active fragment thereof (eg., a CDR-region). Suchfragments are useful as immunosuppressive agents. Alternatively, theantibodies of the invention may have attached to it an effector orreporter molecule. For instance, an antibody or fragment thereof of theinvention may have a macrocycle, for chelating a heavy metal atom, or atoxin, such as ricin, attached to it by a covalent bridging structure.In addition, the Fc fragment or CH₃ domain of a complete antibodymolecule may be replaced or conjugated by an enzyme or toxin molecule,such as chelates, toxins, drugs or prodrugs, and a part of theimmunoglobulin chain may be bonded with a polypeptide effector orreporter molecule, such as biotin, fluorochromes, phosphatases andperoxidases. Bispecific antibodies may also be produced in accordancewith standard procedures well known to those skilled in the art.

The present invention further contemplates genetically modifying theantibody variable and/or constant regions to include effectivelyhomologous variable and constant region amino acid sequences. Generally,changes in the variable region will be made to improve or otherwisemodify antigen binding properties of the antibody or fragment thereof.Changes in the constant region will, in general, be made in order toimprove or otherwise modify biological properties, such as complementfixation, interaction with membranes, and other effector functions.

Typically, the antibodies in accordance with the sixteenth embodiment ofthe invention can be comprised of a polyclonal mixture, or may bemonoclonal in nature. Further, antibodies can be entire immunoglobulinsderived from natural sources, or from recombinant sources. Theantibodies of the present invention may exist in a variety of forms,including for example as a whole antibody, or as an antibody fragment,or other immunologically active fragment thereof, such ascomplementarity determining regions.

Monoclonal antibodies can be obtained by various techniques familiar tothose skilled in the art. For example, spleen cells from an animalimmunised with a desired antigen are immortalised, commonly by fusionwith a myeloma cell, in a manner as described for example, in Kohler andMilstein, Eur. J. Immunol., 6:511–519 (1976), the disclosure of which isincorporated herein by reference.

Alternative methods of immortalisation include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalised cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host. Varioustechniques useful in these arts are discussed, for example, in Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York,(1988), the disclosure of which is incorporated herein by reference,including: immunisation of animals to produce immunoglobulins;production of monoclonal antibodies; labelling immunoglobulins for useas probes; immunoaffinity purification; and immunoassays.

4. An antibody/nucleic acid based method and kit for detecting acidosisactivity

The present invention also encompasses a method of detecting in a samplethe presence of microorganisms involved in lactic acidosis and/or thepotential acid producing characteristics of the microorganisms, whereinthe method comprises:

(a) contacting a sample with the antibody (or fragment thereof) asdefined in accordance with the sixteenth embodiment of the invention,and

(b) detecting the presence of the antibody (or fragment thereof) boundto a microorganism or (fragment thereof) involved in lactic acidosis.

Typically, the method of detecting in a sample the presence ofmicroorganisms involved in lactic acidosis, or potential lactic acidproducing microorganisms may also comprise:

(c) measuring lactic acid present in the digesta or faecal sample ormeasuring pH of said sample; or

(d) measuring amount of lactic acid produced when microorganisms fermentcarbohydrate.

Conditions for incubating an antibody (or fragment thereof) with a testsample vary widely, depending on the format of detection used in theassay, the detection method, and the type and nature of the antibodyused. A person of ordinary skill in the art would readily appreciatethat any one of the commonly available immunological assays could beused in performing the method of detection. For example, these assaysinclude: radio immunoassays, enzyme-linked immunosorbent assays, and/orimmunoflourescent assays.

A kit for performing the above method of the invention contains all thenecessary reagents to carry out the above methods of detection. Forexample, the kit may comprise the following containers:

(a) a first container containing the antibody (or fragment thereof) inaccordance with the sixteenth embodiment of the present invention;

(b) a second container containing a conjugate comprising a bindingpartner of the antibody (or fragment thereof), together with adetectable label.

Typically, the kit may further comprise one or more other containers,containing other components, such as wash reagents, and other reagentscapable of detecting the presence of bound antibodies. More typically,the detection reagents may include: labelled (secondary) antibodies, orwhere the antibody (or fragment thereof) of the present invention isitself labelled, the components may comprise antibody binding reagentscapable of reacting with the labelled antibody (or fragment thereof) ofthe present invention.

Further, the kit of the present invention, as described above inrelation to antibodies, can be readily incorporated, without theexpenditure of inventive ingenuity, into a kit for nucleic acid probes.One skilled in the art would select the nucleic acid probe from thepolynucleotides of the present invention, according to techniques knownin the art as described above. Samples to be tested include but shouldnot be limited to RNA samples of vertebrate tissue.

Such a kit comprises at least one container means having disposedtherein the above-described nucleic acid probe. The kit may furthercomprise other containers comprising one or more of the following: washreagents and reagents capable of detecting the presence of bound nucleicacid probe. Examples of detection reagents include, but are not limitedto radiolabelled probes, enzymatic labelled probes (horseradishperoxidase, alkaline phosphatase), and affinity labelled probes (biotin,avidin, or steptavidin).

In detail, a compartmentalised kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, and like), and containers which contain thereagent detect the hybridised probe, bound antibody, amide product, orthe like.

Furthermore, one skilled in the art would readily recognise that thenucleic acid probes of the present invention can readily be incorporatedinto one of the established kit formats, which are known in the art.

In terms of measuring lactic acid production, the pH of digesta orfaecal material can be measured using colour sensitive reagents or a pHmeter. Lactic acid can be detected using colorimetric techniques usingenzymes, colour reagents and assays assessed with the naked eye or usingspectrometric techniques. Further, identifying potential lactic acidproducing bacteria involves incubation of the sample with carbohydratefollowed by measurement of lactic acid, pH and lactic acid producingbacteria.

The invention will now be described in greater detail by reference tospecific Examples, which should not be construed as in any way limitingon the scope thereof.

EXAMPLES Example 1 Control of S. ruminantium, Clostridium-likevitulinus, S. bovis and S. equinus Material and Methods

Isolation, Identification and characterisation of lactic acid producingbacteria

The following method was used to isolate the most numerous and mostprolific acid-producing bacteria in the gut contents of ruminant andnon-ruminant animals. Rumen fluid samples were obtained via stomach tubefrom sheep and via rumen fistula from cattle 24 h after regular grainfeeding. Faecal samples were obtained directly from the rectum of sheepand cattle and from the freshly voided faeces from horses. Samples wereprocessed for the enumeration of lactic acid bacteria following themethod of Yanke and Cheng (1998) involving one-hour exposure prior toincubation. A semi-selective MRS-agar medium, Oxoid, England (De Man etal. 1960) was modified by adding a freshly prepared reducing solution (1ml containing 0.02 g Cysteine, HCl and 0.026 g of Na₂S.9H₂S.9H₂O per 100ml of media) after boiling then pre-reduced by bubbling with CO₂ on iceuntil cold. The pH of the medium was adjusted to 5.5 during preparation.Viable colonies from roll tubes were picked and incubated into a brothof a basal medium 10 (BM 10) as described by Caldwell and Bryant (1966)with glucose (0.5%) and then again cultured in roll tubes. The procedureof picking colonies and inoculating them into a broth medium followed byinoculating again into roll tubes was repeated twice. At 48 h ofincubation a drop of the broth medium was examined under the microscopeto check the purity of the culture. Morphology and Gram stainingcharacteristics of the isolates were recorded. The ability of culturesto ferment various carbohydrates was evaluated using a broth of BM10with each isolate included at 2 g/L. S. bovis was distinguished from S.equinus on the basis of their ability to ferment starch, insulin andlactose and their ability to survive heating to 60° C. for 30 minuteswas also tested (Hardie 1986).

Fermentation products were measured after 24 h of anaerobic incubationof a broth consisting of BM 10 with glucose or starch (0.5%) at 39° C.At the end of the fermentation period, samples from the media were takenfor measurements of pH, then acidified with sulphuric acid for furtheranalysis of VFA and lactate. VFA concentrations were measured using agas chromatographed (Packard Model 427, Packard Instrument Company,Inc., Illinois, USA), fitted with a Chromsorb ‘W’, acid washed and 60–80mesh column coated with two liquid phases, a: o-phosphoric acid (1.5%w/w) and b: Polypropylene glycol sebacate (17.5% w/w). The temperaturefor the column, detector and the injector was 135, 180 and 210° C.respectively. L-lactate and D-lactate were analyzed by auto-analyzer(Cobas Mir Autoanalyzer, Roche Diagnostics Inc., French Forest, NSW)using an enzymatic procedure (Stat-Pack™ Rapid Lactate Test, Cat No.1112 821, Behring Diagnostics Inc., Somerville, N.J.). The 16S rRNAcomplete gene sequencing and DNA hybridisation techniques (Lane, 1991)were used to identify the most prevalent strains of S. bovis,Clostridium-like vitulinus and S. ruminantium.

Virginiamycin sensitivity test

Two isolates of S. bovis from cattle and sheep (orange pigmented andwhite), five Clostridium-like vitulinus and three isolates of S.ruminantium were incubated in a branch of basal medium 10 with glucose(0.5%) and virginiamycin (VM) at a concentrations of 0, 2, 4, 6 and 8μg/ml. The virginiamycin solution (100 μg VM/ml) was prepared usingEskalin Wettable Powder (WP), 400 g/kg VM (Pfizer Animal Health, NSW,Australia). The WP was dissolved in distilled water (previously boiledand bubbled with nitrogen until cooled) and filter-sterilised whilstbeing gassed with nitrogen. This solution was used immediately afterpreparation. The lowest concentration of VM that resulted in nomeasurable growth of the test bacteria was considered as the MinimumInhibitory Concentration (MIC). The broth-media was inoculated by 0.2 mlof the different fresh viable cultures and 0.1–0.4 ml of VM solution wasadded to each tube to provide the required dose. A sensitivity test wasalso performed when VM was added 3 and 6 h after inoculation, using thesame concentrations of VM. Tests for sensitivity to VM were conductedusing 24 and 4 h h of anaerobic incubation at 39° C.

DNA extraction, PCR amplification and cloning

Freshly grown cultures were withdrawn from screw-cap Hungate tubes byfirst flaming the septum with ethanol. Sub-samples (0.6 ml) of liquidculture were taken with a 25G needle and placed in sterile 1.5-mLcentrifuge tubes. Cells were harvested as a pellet followingcentrifugation at 13500 g for 2 min. The supernatant was poured off andthe cells resuspended in approximately 30 μL of culture fluid remaining.

DNA was extracted form the concentrated cell suspension using a Fast DNASPIN KIT (BIO 101, Inc, CA). Extracted DNA was visualized on a 1% TAEgel amended with 0.5 μl of 10 mg/kg ethidium bromide per 50 mL agarose.PCR was used to amplify 16S rDNAs in 100 μL reactions. Each reactiontube contained 200 ng of each primer, 10 μl of 10× buffer, 6 μL ofMgCl₂, 1 U of Tth DNA polymerase (Biotech International, Perth,Australia), 10 μL of 4×0.5 mM dNTP's, and the remainder made up withsterile Milli-Q water and 2 μL concentrated cell suspension or DNAextract. Reactions were overlaid with sterile mineral oil and carriedout in a thermocycler (Perkin-Elmer DNA Thermal Cycler 480).Thermocycling parameters employed after at 96° C., denaturation for 10min were 28 cycles of 1 min at 94° C., 1 min at an annealingtemperature, and 2 min at 72° C. A further extension step involving 1min at 48° C. and 5 min at 72° C. was also employed. The primers usedwere 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) (SEQ ID No:8) and 1492r(5′-GGTTACCTTGTTACGACT-3′) (SEQ ID No;9) (Lane 1991). In some casesGeneReleaser (Bioventures Inc., Tennessee, USA) was used according tothe manufacturer's instructions in the reactions outlined above. PCRproducts were purified using a QIAquick PCR purification kit (QIAGEN,Victoria Australia) according to the manufacturer's instructions.

Sequencing of 16S rDNA

All 16S rDNA samples were initially partially sequenced using theuniversal 16S rRNA primer, 530f (5′-GTGCCAGCMGCCGCCG-3′) (SEQ ID No:10)and an ABI Big Dye Terminator Cycle Sequencing Ready Reaction Mix kit(Victoria, Australia). Selected 16S rDNA were subsequently fullysequenced on both strands using the following primers: 519r(5′-GWATTACCGCGGCKGCTG-3′) (SEQ ID No:11), 27f, 907r(5′-CCGTCAATTCMTTTRAGTTT-3′) (DEQ ID No:12), 926f(5′AAACTYAAAKGAATTGACGG-3′) (SEQ ID No:13).

Approximately 100 ng of purified PCR product and 25 ng of primer wereused in the sequencing reactions. Thermal cycling was carried out in anMJ Research PTC-100 thermocycler with an initial denaturation step of96° C. for 2 min, following by 25 cycles of 50° C. for 15s, 60° C. for 4min, and 96° C. for 30s. The resulting cycle sequencing products werepurified using the ethanol plus sodium acetate method (ABI, Australia).Purified sequencing products were submitted to the Australian GenomeResearch Facility for analysis on an Applied Biosystems 377 automatedsequencer.

Phylogenetic analysis

Phylogenetic analysis of 16S rDNA's was carried out according to Dojkaet al. (1998). Briefly, sequences were aligned and compiled in SeqEd(Applied Biosystems Australia). Compiled sequences were compared withthose on publicly available databases by use of the BLAST (BasicAlignment Search Tool) (Altschul et al. 1990) to determine approximatephylogenetic affiliations. Compiled sequences were then aligned usingthe ARB software package (Strunk et al. unpublished) and refinedmanually. Phylogenetic trees based on comparative analysis of the 16SrRNA genes were construction by performing evolution distance analyseson these alignments using the appropriate tool in the ARB database. Therobustness of the tree topology was tested by performing bootstrapping(2000 replicates) in PAUP test version 4.0d65.

Results

All twenty streptococcal isolates (6 from sheep, 2 from cattle and 12from horses) were 99% identical in sequence to each other and to S.bovis and S. equinus. These isolates therefore belong to the genusStreptococcus and are members of either S. bovis or S. equinus. Three ofthe 12 streptococcal isolates from horses were identified, as S. equinusas they did not ferment starch or insulin and only one of them fermentedlactose.

Sb R1 was the dominant S. bovis strain across all three animal species(cattle, sheep and horse). It grows into bright orange-centered colonieson MRS agar roll tubes and produces an orange pigment in a broth of BM10 with glucose or starch. The cells are 0.9–1.0 μm in diameter andoften encapsulated, occurred mainly in pairs, short chains of 4–10 cellsand singles. The doubling time for S. bovis was estimated to be 24minutes. Three streptococcal isolates (SE R1, SE R2 & SE R3) from horseswere identified as S. equinus. The cells occurred mainly in long chains.

Table 1 shows the fermentation end products of S. bovis, S. equinus,Clostridium-like vitulinus and S. ruminantium isolates. All of the S.bovis bacteria could ferment cellobiose, fructose, galactose, glucose,lactose, maltose, mannose, raffinose, starch and sucrose, but notarabinose, glycerol, mannitol, ribose, sorbitol or xylose and two of thewhite pigments isolates did not ferment insulin (Table 2). L-lactate wasthe main fermentation product of S. bovis and S. equinus.

The Clostridium-like vitulinus-like isolates have 100% sequence identityto each other, and 97% identity with Clostridium-like vitulinus.Clostridium-like vitulinus cells occurred in different shapes and sizes,mainly straight rods in singles, pairs and short chains and some cellshave the tendency to branch. Clostridium-like vitulinus isolates couldnot ferment starch or xylose but grew on cellobiose, fructose, glucose,mannose, raffinose and sucrose (Table 2). Two Clostridium-like vitulinusisolates produced D-lactate and one produced L- and D-lactate at equalproportions.

S. ruminantium was isolated from the rumen of grass-adapted sheep thatreceived a grain supplement with or without virginiamycin. The threeSelenomonas-like isolates formed two distinct lines of descent: Type A(isolate SR R1); and Type B (isolate SR R2 and isolate SR R3). Type Ahas >97% identity to S. ruminantium. This indicates that all threeisolates are members of the genus Selenomonas and Type A belongs to thespecies S. ruminantium. One isolate (SR R1) produced L-Lactate while theother two (SR R2 and SR R3) produced L- and D-lactate (Table 3). Adifference between SR R2 and SR R3 was that SR R3 produced 42.5% morelactic acid from starch than SR R2. S. ruminantium grew on mostcarbohydrate sources (Table 2) but their growth was noticeably high onglucose and sucrose compared with other sources and only one isolate (SRR1) did not ferment starch. The cells are 0.5–0.7×1.5–3.0 μm, occurredmainly in single short crescent rods, with round ends and Gram-negative.Similar amounts of L-lactate were produced from glucose, starch orraftilose by the different S. bovis isolates (Table 4).

Incubation in vitro of S. bovis, Clostridium-like vitulinus and S.ruminantium isolates in a broth of Basal Medium 10 with glucose (0.5%)with different concentration of VM (0, 2, 4, 6 and 8 μg/ml) clearlydemonstrated sensitivity of S. bovis to virginiamycin. Clostridium-likeSelenomonas isolates (Table 5) showed varying levels of resistance toVM. LV R1 and LV R5 were very sensitivity to VM with a MIC level of 2μg/ml. Isolates LV R2 and LV R3 resisted levels up to 4 μg/ml and LV R4up to 6 μg/ml. S. ruminantium isolates were resistant to the highest VMlevels (8 μg/ml). Sensitivity of Clostridium-like bacteria to VM wasdifferent when VM was added at 3 and 6 h after inoculation. AllClostridium-like cultures growing for 3 h before the addition of VM hadhigher MIC than when VM was added to immediately after inoculation.Sensitivity to VM was also lower when assessed using a 48 h incubation.All Clostridium-like isolates were resistant to the highest VM levels (8μg/ml) when cultures were allowed to grow for 6 h before the addition ofVM.

Discussion

The presence of Clostridium-like vitulinus and S. ruminantium in rumenfluid and the faeces of sheep fed diets with or without VM demonstratedthe resistance of some Clostridium-like strains to viginiamycin (VM).This resistance of Clostridium-like and Selenomonas isolates has beenconfirmed by results from anaerobic in vitro incubation of in a broth ofBM 10 with glucose (0.5%) with or without VM.

A sensitivity test showed that S. bovis strains are quite sensitive toVM and this reaction seems to be irreversible and fatal. On the otherhand the reaction of Clostridium-like cells to VM with may be reversibleone and the initial reaction results in only bacteriostasis rather thanbactericidal effects.

The fact that the Gram-negative bacteria S. ruminantium i snot sensitiveto VM suggests that there will be situations where are VM may notprovide for protection against acidosis. Godfrey et al. (1995) andNagaraja et al. (1995) reported that in grain-adapted sheep theinclusion of VM with grain did not prevent decreased ruminal pH andincreased lactate. This may indicate that under conditions whereClostridium-like bacteria and Selenomonas are well established in thegut, the use of VM may not be as effective in controlling acidosis.

These results also indicate why vaccination against S. bovis alone isunlikely to effectively control lactic acid accumulation when animalsconsume high levels of readily fermentable carbohydrates. Under theseare conditions Selenomonas and Clostridium-like will continue to producelactic acid even if the activity of S. bovis is inhibited.

It is clear that any diagnosis, vaccine or other method to prevent orcontrol fermentative acidosis should detect and or be active against atleast S. ruminantium, Clostridium-like vitulinus, S. bovis and S.equinus.

Example 2

Samples of faceces were collected from 12 horses fed a mixture ofchopped leucerne hay and different types of cereal grain (oats, barley,triticale and sorghum). The dominant lactic acid producing bacteria wereisolated using the same method as described in Example 1. Two bacteriawere isolated: Streptococcus bovis (SbR1) and Streptococcus equinus(SER1 and SER2). Based on these results it is important that anyvaccine, diagnosis or other treatment to prevent or control acidosisshould be effective against both S. bovis and S. equinus. Under someconditions it will also be necessary for these diagnostic tools andmethods of treatment to be effective against S. ruminantium,Clostridium-like vitulinus as well as S. bovis and S. equinus.

In certain conditions it is likely that Selenomonas ruminantium andClostridium-like vitulinus bacteria may be present and in this caseanimals should be vaccinated against both Gram-positive andGram-negative lactic acid producing bacteria.

Example 3

Following calving, cows grazing lush green pasture are exposed to highlevels of soluble carbohydrate in the form of fructans in grasses andsugars and starch in clovers. In addition to fermentable carbohydratesin the pasture, concentrate feed supplements, based on cereal grain, arefed twice daily during milking. The fructants in pastures and the starchin legumes and concentrate are rapidly fermented in either the rumen orthe hind gut to form a range of volatile fatty acids and lactic acid.The accumulation of acids in the gut contribute to the metabolic acidload of the animal, can cause inflammation of the gut wall leading tostimulation of the immune system and can lead to increased pathogenicityin the populations of bacteria and parasites within the gut. The adverseeffects of acid accumulation in the gut result in reduced productivityand an increased incidences of disease including lameness, respiratoryconditions and mastitis.

Acid accumulation in the gut under this dietary regime can besatisfactorily reduced by controlling two of the principle acidproducing bacteria Selenomonas ruminantium and Streptococcus spp. As oneof these bacteria is Gram-negative and the other Gram-positive twoapproaches are used. A vaccine is used to control Selenomonasruminantium and the feed additive virginiamycin is used to controlStreptococcus spp. The vaccine is prepared by combining washed bacteriaof the isolate Selenomonas ruminantium (SRR1) (between 10⁷ and 10¹¹cells/ml) and 1 ml of the adjvuant DEAE-Dextran. This is injectedintramuscularly into dairy cattle prior to calving in order to developantibodies against Selenomonas ruminantium lactic acid producingbacteria. The feed additive virginiamycin is included in the concentratefeed to provide between 150 and 450 mg per head per day.

Example 4

Pigs are often fed diets containing high concentrations of starch andsome of this starch can pass undigested to the hind gut where itundergoes rapid fermentation leading to the accumulation of tactic acid.The adverse effects of lactic acid accumulation and low pH in the hindgut of pigs includes metabolic acidosis, compromised integrity of thegut wall and increased pathogenicity of gut organisms. Piglets can betreated against this condition by immunising them againstPrevotella-like bacteria such as LAB01/07-3, or Clostridium-likevitulinus and can be further protected by the inclusion of an antibioticfeed additive in the diet such as virginiamycin.

Example 5

Dogs and cats have evolved as carnivorous species but are not fed dietscontaining processed cereal grain. The carbohydrate fraction of the dietis poorly digested in the small intestine of these animals. Undigestedcarbohydrate passing from the small intestine to the hind gut is rapidlyfermented and result in accumulation of acid. The accumulation of acidand the subsequent adverse side effects can be prevented by vaccinationagainst Prevotella-like (LAB03/D35). Enterococcus-like (LAB05/D23),Clostridium-like and/or Selenomonas ruminantium. Further protectionagainst lactic acid production by Streptococcus spp. can be achievedusing virginiamycin or similar type of Gram-positive antibiotic compoundin the feed.

Example 6

Horses grazing lush green feed and/or supplemented with cereal grainsare at risk of incomplete carbohydrates digestion in the small intestineand fermentative lactic acid accumulation in the hind gut. Acidaccumulation in the gut can result in laminitis an development ofadverse behaviour. The risk of these potential problems can be reducedby immunisation against Streptococcus equinus and the strategic use ofantibiotics active against Clostridium-like virtulinus and Selenomonasruminantium.

Example 7

Humans suffering from lactose intolerance, irritable bowel syndrome, orany side effects of acidic gut syndrome can be immunised againstClostridium-like virtulinus and/or Selenomonas ruminantium and treatedstrategically with antibiotics such as virginiamycin and problocks suchas Megasphera elsdeni,i Bifidabacteria, Lu12 or Su109.

Example 8

1. Seroconversion in sheep vaccinated with bacterin comprised offormalin-killed lactic acid producing bacteria.

An experiment was conducted to examine whether sheep would raiseantibodies in response to a range of bacterins comprising bacteria thatare commensal organisms in the gatrointestinal tract of ruminants orhorses.

Materials and Methods

Forty nine adult Merino whether( approximately three years of age werechecked for uniformity and physical appearance before entering thestudy. All animals were weighed and treated against internal parasites(ivermectin) approximately 3 weeks before being randomly assigned to oneof seven experimental groups (seven animals per treatment group). Eachanimal received a different vaccination treatment consisting of one ofthe bacterins described in Table 8.1 and animals in the control groupreceived adjuvant only. All of the sheep remained at pressure for 34weeks before being housed for a grain challenge experiment. During thegrazing phase of the experiment the sheep were given access to goodquality green pasture whenever possible.

TABLE 8.1 Details of microorganisms used in vaccine preparation Expt. IDAccession Date of Microbe code No. Deposit 1. Streptococcus bovis Sb5N94/8255 Mar. 8, 1994 2 Streptococcus bovis A2 NM99/04455 Jun. 24, 19993 Streptococcus equinus Seq NM99/04457 Jun. 24, 1999 4 Clostridium-likevitulinus LV NM99/04462 Jun. 24, 1999 5 Selenomonas ruminantium SR1NM99/04458 Jun. 24, 1999 6 Selenomonas ruminantium SR2 NM99/04460 Jun.24, 1999Preparation of vaccines: Frozen isolates of all bacteria were thawedthen cultured in BM 10 for between 6 and 10 hours. All incubation wereat 37° C. Formalin was added to the incubation flasks to achieve a finalconcentration of 0.5% in the final solution. The cells ere thenharvested by centrifugation and washed 3 times using sterile PBS beforebeing made to final volume in PBS with a concentration of 5×10⁹cells/ml.

The vaccines were prepared by mixing equal volumes of the bacterialsuspension and Freund's complete adjuvant in the case of the primaryvaccinations and Freund's incomplete adjuvant for booster injections.The control animals received sterile PBS and adjvuant. Even vaccinationinjection was 2 ml administered intramuscularly. For the primary andsecondary vaccination, each sheep received 4 injections, one in eachshoulder and 1 into each of the semi-membranosa muscles of each hindleg. The booster given in the 35^(th) week after the primary injectionwas only administered to the two hind legs.

Samples of blood were taken according to the schedule in Table 8.2 formeasurements of antibodies against the bacteria use to vaccinate eachtreatment group. Development of ELISA methods for measurement ofrelevant antibody titres was an important component of this study and isdescribed below.

ELISA protocol: Bacterial antigen was prepared in the same way asdescribed for the immunisations, and stored in aliquots at −20° C.Optimal antigen dilution was found to be 1:260 (diluted in carbonatebuffer); 100 μl of this suspension was added to each well of ELISAplates (Immunolon II flat bottom 96-well plates). Plates were thencovered and incubated overnight at 4° C. Antigen was removed from thewells and the plates washed 3 times in PBST (NaCl & 8 g, Na2HPO4 1.15 g,KH2PO4 0.2 g, 0.5 ml Tween 20, in 1000 ml MQ water, pH 7.2–7.4) using anautomated plate washer (TiterTek Microplate Washer 120, FlowLaboratories. Blocking buffer (1% Bovine serum albumin (Sigma) in PBST)was added to each well and allowed to incubate at 37° C. for 1 hour,followed by 3 washes and PBST. Titrations of the test sera wereperformed and optimal dilutions for a standard assay determined. Testsera were analysed by diluting sera in blocking buffer at levels foundto be optimal for each antigen. Following dilution 100 μl of each testserum was analysed in duplicate wells; in addition, pre-vaccinationserum was always included in each plate. Negative controls (PBS) andstandards were included in triplicate. Standard were prepared by doubleserial dilution (in blocking buffer) starting at 1:100 ofpreviously-assayed sheep sera known to contain Sb5 antibodies. Afteradding the serum samples, plates were incubated for 1 hour at 37° C.followed by 3 washes in PBST. The optimal dilution of the horseradishperoxidase conjugates was found to be 1:1,000 for the sheep (rabbitanti-shape-IgG (H & L) HRP, BioRad),. Conjugates were dilutedaccordingly in blocking buffer, 100 μl added to appropriate wells, andthe plates incubated for 1 hour at 37° C. Plates were again washed 3times with PBST, and 100 μl of substrate added (34 mg o-phenylenediamnein 100 ml citriate phosphate buffer (light sensitive), 50 μl of 30% H₂O₂(added just prior use) (citrate phosphate buffer: citric acid 7.3 g.Na₂HPO₄ 9.5 g, 800 ml MQ water, pH to 6 with 10 M NaOH and brought to1000 ml). Plates were incubated in the dark at room temperature for 30mins at which time the enzymatic reaction was stopped by the addition of100 μl of 2N sulphuric acid. Absorbances were read in a microplatereader (Benchmark, BioRad) and the data analysed using MicroplateManager/PC 4.0 (BioRad) software. Analysis parameters and standard curveinformation were included for each plate.

Samples of rumen fluid were taken via stomach tube and pH was measuredbefore taking a sub-sample for determination of potential lactic acidproduction. The remainder of the sample was acidified for latermeasurement of volatile fatty acid concentrations. Potential lactic acidproduction was measured by incubating 4 ml of rumen fluid with 1 mlglucose solution containing 50 mg glucose/ml. Following incubation withglucose for 20 hours pH was measured before acidification with 0.1 ml ofconcentrated sulphuric acid and frozen for subsequent analysis of lacticacid concentration.

TABLE 8.2 Schedule of immunisation and sampling to monitor systemicantbody levels and the consequences of vaccination in Example 8 WeekProcedure −1 Blood sample (First pro-bleed) 0 Blood sample (Secondpre-bleed) + primary immunisation 2 Blood sample for antibodymeasurement 4 Blood sample + secondary (first booster) immunisation 6Blood sample + collect rumen fluid and faeces 8 Blood sample 10 Bloodsample + collect rumen fluid and faeces 12 Blood sample 14 Blood sample

For bacterial enumeration a rumen fluid samples were processed followingthe one-hour exposure method prior to incubation of Yanke and Cheng(1998). The rumen fluid was serially diluted ten-fold in anaerobicdilution solution (ADS) (Caldwell and Bryant, 1966) to a final dilutionof 10⁻⁸. Three dilutions (10⁻⁶, 10⁻⁷ and 10⁻⁸) were used to inoculatethe media roll tubes in triplicate for each dilution. A modifiedsemi-selective MRS-Agar medium, Oxoid, England (de Man et al., 1960) wasused to culture the lactic acid bacteria was the pH of the mediumadjusted to 5.5. The tubes were incubated at 39° C. for three days.Faecal samples collected directly from the rectum were mixed with twotimes their weight of distilled water. 4 ml of the slurry was incubatedwith glucose solution as described for rumen fluid potential lactic acidmeasurement. A further quantity (approx 10 ml) was filtered through 4layers of cheese cloth before measuring pH and then acidifying thesample for later analysis of volatile fatty acids.

VFA concentrations measured by gas chromatography (Hewlett-Packard) andL(+)lactam and D(−) lactate analyzed by auto-analyzer (Cobas MiraAutoanalyser, Roche Diagnostics Inc., French Forest, NSW) using anenzymatic procedure (Stat-Pak™ Rapid Lactate Test, Behring DiagnosticInc., Somerville, N.J.)

Results

There were good levels of seroconversion (Table 8.3) in response to thevarious bacteria used in this experiment and elevated antibody titrespersisted to week 14 following the primary vaccination.

TABLE 8.3 Antibody titres (Ab units × 1000) measured in serum from sheepimmunised with different bacterin. Time in weeks refers to weeks afterthe primary vaccination. All sheep were re-immumised 4 weeks after theprimary vaccination. Streptococcus bovis; Streptococcus equinus;Clostridium-like vitulinus; Selenomonas ruminantium isolates R1 and R2respectively Group Sheep Bacterin Pre vacc'n Week 2 Week 4 Week 6 Week 8Week 10 Week 12 Week 14 1 1 S. bovis Sb5 0 0 0 240 0 166 271 807 1 3 S.bovis Sb5 0 0 0 0 0 0 0 295 1 5 S. bovis Sb5 0 0 0 462 224 700 381 0 1 6S. bovis Sb5 0 114 183 557 224 370 403 386 1 7 S. bovis Sb5 88 178 2961,136 620 490 588 538 2 8 S. bovis A2 0 28 70 311 100 294 712 528 2 9 S.bovis A2 0 0 25 181 116 232 2,714 712 2 10 S. bovis A2 11 14 37 181 218187 1,094 498 2 11 S. bovis A2 0 8 25 121 175 329 3,567 3,567 2 12 S.bovis A2 9 24 25 120 81 79 1,823 1,321 2 13 S. bovis A2 6 11 15 64 99110 1,823 2,904 2 14 S. bovis A2 12 33 44 197 227 180 3,567 2,220 3 15S. equinus 9 22 85 105,000 2,011 5,197 10,140 5,622 3 16 S. equinus 1011 20 302 80 38 23 18 3 17 S. equinus 8 9 11 484 558 871 3,657 3,936 318 S. equinus 9 28 123 300,000 18,710 27,010 265,900 73,440 3 19 S.equinus 9 28 161 6,567 4,430 971 385 784 3 20 S. equinus 9 20 53 2,225191 824 501 1,250 3 21 S. equinus 9 13 35 797 109 90 67 101 4 22 C.vitulinus 13 206 98 883 477 899 324 1,221 4 23 C. vitulinus 33 115 142394 244 265 100 89 4 24 C. vitulinus 1,014 202 265 103 130 43 45 554 425 C. vitulinus 127 145 365 1,181 477 394 365 159 4 26 C. vitulinus 0 0313 2,948 243 634 313 234 4 27 C. vitulinus 0 483 292 1,944 668 1,1972,898 1,446 4 28 C. vitulinus 0 243 417 123 469 602 243 0 5 29 SR R1 17125 120 181 246 1,346 829 688 5 30 SR R1 16 446 172 229 303 392 441 3965 31 SR R1 2 261 300 261 114 89 648 441 5 32 SR R1 15 145 115 545 16,1305,058 40,660 24,140 5 33 SR R1 21 159 124 241 145 124 127 92 5 34 SR R10 55 46 132 132 67 86 83 5 35 SR R1 0 83 67 109 94 170 384 563 6 36 SrR2 0 117 3,292 3,403 2,570 9,975 4,367 3,132 6 37 Sr R2 7 14 49 110 261227 249 351 6 38 Sr R2 6 12 63 329 619 430 1,156 512 6 39 Sr R2 6 51 110210 169 261 437 1,450 6 40 Sr R2 8 23 78 723 913 12,000 3,240 1,174 6 41Sr R2 10 19 72 107 185 322 88 124 6 42 Sr R2 8 30 2,255 11,940 432 235214 442

During the period that sheep were grazing spring and summer pasturesthere was no significant effect of vaccination treatment on faecal drymatter content, as an index of faecal consistency and diarrhoea (seeTable 8.4). Although not statistically significant there were morelactic acid bacteria (growing on MRS medium) to the rumen fluid of sheepimmunized against the S. ruminantium bacteria. This is an importantresult and is consistent with the pH of rumen fluid shown in Table 8.5(week 10) and Table 8.6 (week 38) where the treatment groups withhighest numbers of lactic acid producers were found to have lowest pH.

TABLE 8.4 Body weight of sheep at week 34 and the faecal dry matter %measured during weeks. Body Body weight Faecal dry Faecal DM Faecal dryFaecal DM weight Week 34 matter Week 0 matter Weeks 6 & 10 (Week 34) SE(Week 0) SE (Weeks 6 & 10) SE Sb5 56.2 2.87 23.7 1.86 25.0 1.617 A2 52.92.42 21.4 1.93 22.5 1.810 Seq 54.0 0.75 23.8 2.69 22.1 1.760 LV 56.01.54 22.4 1.36 22.3 1.803 SR1 54.4 1.83 24.8 1.36 22.5 1.803 SR2 56.31.83 24.7 2.13 24.2 1.393 Control 56.3 1.44 26.4 2.63 22.6 0.998

TABLE 8.5 Rumen and faecal pH and the pH following incubation withglucose in samples taken from vaccinated sheep 6 and 10 weeks followingvaccination with a range of lactic acid-producing bacterins. Numbers ofbacterias (cells/ml) measured in the rumen fluid during week 10. LacticLactic Ru- Fae- Total acid acid men cal bacteria × bacteria × bacteriapH SE pH SE 10⁸ 10⁷ % of total Sb5 7.0 0.075 7.7 0.77 10.1 8.3 9.1 A27.0 0.075 7.4 0.77 1.5 9.2 6.2 S.Eq 6.9 0.075 7.6 0.77 1.3 6.7 5.4 LV7.0 0.075 7.6 0.77 1.3 6.8 5.7 SR R1 6.9 0.075 7.7 0.77 2.1 46.5 12.7 SRR2 6.9 0.075 7.5 0.77 2.8 22.0 6.3 Control 6.9 0.078 4.5 0.80 1.3 11.97.92. Response to grain acidosis challenge in sheep 38 weeks after initialimmunisation

Thirty four weeks after the primary vaccination all sheep were broughtinto the animal house where they were weighted and housed in individualpens with free access to clean water. All animals were treated with abroad-spectrum anthelmintic. For a period of four weeks animals were feda mixture of cereal & lucerne chaff (1100 g/d).

Five days after entering the shed sheep were re-vaccinated as describedabove and were given approximately 60 g wheat/d together with 1100 gchaff. During week 37 (post primary vaccination) samples of rumen fluidand faeces were taken for measurement of pH and potential lactateproduction. Portion of the samples were also taken for later analysis ofvolatile fatty acids.

Grain challenge and sampling: On day 1 of the challenge, 1 kg of wholewheat was offered to each sheep at approximately7 AM. For those sheepthat had not eaten all the wheat within 2 hours an amount of crackedwheat, equivalent to the residue and was administered into the rumen viastomach tube as a slurry made up with water. Immediately afteradministration of the wheat, blood samples were taken for antibodymeasurements and for haptoglobin determination. Approximately eighthours after first feeding the wheat (4 PM), samples of rumen fluid weretaken for measurement of pH, VFA and lactate.

On the following morning a rumen sample was taken for measurement of pH,VFA and lactate and a check made of faecal consistency (faecal scores).Faecal samples were collected for measurement of pH and dry matter. Allsheep were then offered 500 g wheat and 1 hour later refusals weighed.An amount of cracked wheat equivalent to the residue and administeredinto the rumen via a stomach tube. Chaffed lucerne and cereal hay wasfed ad libitum after the final dose of wheat and intakes were measureddaily. Samples of rumen fluid were taken 8 hour later (32 h from firstwheat) and faeces were scored for consistency and sampled for pH. Fortyeight hours after first offering wheat to the sheep of a final rumensample was taken and faecal scores were recorded. A sample of blood wastaken for haptoglobin analysis.

Antibody levels, rumen pH and the potential lactic acid productionmeasured during week 38 after primary challenge (4 weeks after thesecond booster injection administered during week 34) are summarised inTable 8.6. The major finding is that the rumen pH pre-grain challengeand on “normal” feed was significantly (P<0.001) affected by vaccinationtreatment. The control animals had the highest level of acid and thegroup vaccinated with Sb A2 had the lowest acidity (highest pH). Thesedata strongly suggest an affect on vaccination on acid production duringrumen fermentation on normal diets.

In vivo responses to the “intake” of excessive levels of wheat aresummarised in Table 8.7. There were significance (P<0.01) differences inrumen lactic acid concentrations with the sheep vaccinated with Sb A2,vitulinus (LV) and SR2 having the lowest concentrations of lactic acidin the rumen.

Faecal consistency scores were significantly (p<0.01) different in sheepvaccinated with different antigens. The sheep vaccinated against S.equinus consistency had more diarrhoea than the other treatment groupsand these sheep also had the lowest feed intake.

TABLE 8.6 Antibody titres (Ab units × 1000) at week 38, rumen fluid pHand potential lactate production measured in rumen fluid and faeces.Potential Potential Antibody Rumen lactate lactate titre fluid pH RumenFaeces Treatment Week 38 Week 38 Week 38 Week 38 Sb5 260 7.02 27.3 18.9A2 30 7.09 19.1 25.2 Seq 15 6.95 18.6 24.7 LV 3492 6.90 16.8 19.7 SR1496 6.75 23.4 28.8 SR2 690 6.82 13.6 19.1 control 0* 6.89 30.6 27.2 Sig0.001 0.001 NS NS SE 314 0.078 6.0 3.5 *Animals in the control group andantibodies against LV at a level <2% of the group vaccinated against LV,and no antibodies against other antigens used in this experiment.

TABLE 8.7 Responses in vaccinated sheep to grain acidosis challenge.Faecal consistency score were based on 1 = normal pelleted faeces and 5= diarrhoea. Feed intake values are: g/d. Average Average faecal feedRumen Rumen Faecal score intake lactate pH pH 24 to 48 for 72 h (32 hafter (32 h after (24 h after h after after Treatment wheat) wheat)wheat) first wheat first wheat Sb5 25.5 5.81 6.51 1.73 674 A2 0.6 5.995.54 3.31 639 Seq 56.3 5.65 5.90 3.73 516 LV 0.0 5.91 5.48 2.77 599 SR119.3 5.83 6.19 2.25 665 SR2 2.1 5.83 5.67 3.39 697 Control 0.1 6.16 5.752.31 786 Sig 0.01 NS NS 0.01 NS SE 12.7 2.1 0.45 0.38  69Discussion

The results of this study have shown that it is possible to immunisesheep against aggressive lactic acid producing bacteria that normallyinhabit the gastrointestinal tract and that by producing antibodies tothese lactic acid producing bacteria the animal is able to alter theresponse to dietary carbohydrate overload. Changes in response to thegrain overload challenge include reduced amounts of rumen lactic acidaccumulation in sheep vaccinated with A2. SR2 and Cv. Sheep in thesetreatment groups had the lowest levels of lactic acid of the vaccinatedsheep. The treatment group with the highest rumen lactic acid (S.equinus) had the lowest subsequent feed intake and the most serverdiarrhoea.

It is concluded that lactic acid bacteria other than Sb 5 can be used asantigens to alter the animals ability to respond to fermentativeacidosis challenge and that other bacteria such as A2, Cv and SR2 mayactually be more effective antigens in preparing the animal to controllactic acid build up in the rumen following a carbohydrate challenge.The only bacterial antigen used in this study that was not isolated froma ruminant animal (S. equinus, isolated from a horse) had the leasteffect in reducing lactic acid accumulation. This indicates theimportance of discovering and isolating the lactic acid producingbacteria specifically associated with type of animal and relevant diet.

Example 9 Isolation of Predominant and Aggressive Lactic Acid ProducingBacteria from the Digestive Tract of Pigs, Dogs and Humans

The lactic acid producing bacteria in the hind gut or large bowel(caecum, colon and rectum) of different vertebrate species varydepending on the substrate available for fermentation and the rate ofturnover of digesta in the large bowel as this determines averageresidence time of bacteria in the large bowel. The amount and nature ofsubstrate entering the large bowel is determined by two major factors:diet and the pre-digestion of dietary substrates in the stomach andsmall intestine prior to passage of digesta into the large bowel.Pre-digestion in the stomach and small intestine are, in turn, areinfluenced by the nature and efficiency of digestive enzymes (such asamylase, amyloglucosidase, pepsin, pepsinogen, brush—borer carbohydratedegrading enzyme), rate of intake and effective of chewing and particlesize diminution such as occurs in the crop of bird, the rate of digestapassage (determined by factors such as animal species, meal size, levelof stress) and the absorptive capacity of the digestive tract. The rateof turnover of digesta in the large bowel is determined by the size ofcompartments such as the caecum and colon relative to the flow ofdigests through the compartments. The half-time for digesta residence indifferent animal species can vary from days to just several hours.Variation in residence time and the amount and nature of substrateavailable for fermentation in different vertebrate species, consumingdifferent diets, therefore means that there will be importantdifferences in the dominant lactic acid producing bacteria that colonisethe large bowel.

Even in situations where it is well known that a species of bacteriasuch as the lactobilli or streptococci produce lactic acid it isessential to discover and isolate the actual strains of that genusspecifically capable of surviving in the particular digestivecompartment under the prevailing conditions of flow and substrateavailability with the ability to compete with other gut bacteria.Moreover it is essential to identify and select the strain(s)numerically important and capable of abundant lactic acid production.The experiments described below were designed to discover and isolatethe predominant lactic acid producing bacteria from the digestive tractof pigs, dogs and humans. Since there are no major physiological changesin the pH, temperature or anaerobic/aerobic status of digesta duringpassage from the caecum through the colon and rectum, samples of faeceswere considered to provide suitable material for isolating the lacticacid bacteria of the large bowel of the respective vertebrate species.

(i) Discovery and isolation of lactic acid producing bacteria from pigs

Commercial diets for pig production are commonly based on cereal grain.The grain is normally processed by grinding through a hammer mill beforemixing with protein, vitamins and minerals to form a complete diet. Inthe current study samples of faeces were taken from 5 pigs in acommercial piggery. The pigs weighing around 40 kg and 6 weeks of agewere housed in group pens and were considered to be in good health withno signs of abnormality at the time of sampling. The diet containedbarley and sorghum (50:50 by weight) as the source of grain and wasformulated to contain 14% protein. The level of feeding was designatedto be ad libitum and was offered to the animals twice per day.

Faeces were prepared as described in Example 1 for the discovery andisolation of the most numerous species/strains of lactic acid bacteria.The process of selecting colonies for dominance and lactic acidproduction and isolating pure strains involved enumeration of lacticacid strains on a modified semi selective MRS and growth in a broth ofglucose based BM 10 as described in Example 1. Those isolates consideredthe most important lactic acid producers were studied microscopicallyfor purity and gross morphological classification.

There were 2 isolates considered to be different from lactic acidproducing microbes isolated in our previous presearch. These isolatesare described in Table 9.1 and have been deposited with the AustralianGovernment Analytical Laboratories (AGAL). Dates and accession numbersare summarised in Table 9.1

TABLE 9.1 Details of two lactic acid-producing producing bacterialisolates discovered in the faeces of pigs under commercial piggeryconditions. Lab code number LAB 01/07-3 LAB 02/11-2 Description Shortrods arranged Short straight rods in short chains and with round ends,also filaments, Gram+ long thin rods Preliminary Prevotella-likenon-slime producing classification lactic acid bacterial isolates AGALNMOO/12630 NM00/12631 accession number Date of AGAL Jun. 29, 2000 Jun.29, 2000 deposition(ii) Discovery and isolation of lactic acid producing bacteria from dogs

Two experiments were undertaken to identify the most important lacticacid bacteria in the large bowel of dogs on a high carbohydrate dies fedwith, or without, the antibiotic feed additive virginiamycin. Asindicated in Example 1 there are situations where the antibiotic feedadditive virginiamycin produces incomplete control over lactic acidaccumulation in the digestive tract of ruminant animals. The experimentoutlined below was undertaken to determine the level of control ofvirginiamycin over lactic acid accumulation in the large bowel of dogs.The second experiment in this Example 9 was designed to discover andisolate the dominant lactic acid, producing bacteria from the faeces ofdogs fed the same diet either with or without virginiamycin.

1. Dose virginiamycin control lactic acid production in the large bowelof dogs?

The main objective of this trial was to investigate various doses ofvirginiamycin in terms of its effect on faecal consistency and hind gutfermentation.

Design: There were 4 treatment groups consisting of unmedicated control,and virginiamycin at 3 levels: 0.25, 0.5 and 1 mg/kg body weight. Dogswere acclimatised to a dict based on the tinned ‘No Frills’ dog food forapproximately 2 weeks prior to a seven-day treatment period in whichfructo-oligosaccharide raftilose was added to the diet at a rate of 3.5g/kg metabolic body weight. There were five dogs per treatment.

Measurements were made of faecal pH, dry matter, consistency andchemical assays were conducted to measure VFA, D-lactic acid andL-lactic acid.

Results The main results are summarised in Table 9.2.

-   -   The effect of raftilose inclusion is to reduce pH by        approximately 1 unit. The secondary effects associated with the        increased acidity include more sloppy consistency, reduced dry        matter and increased lactic acid.    -   There was little effect of the virginiamycin on the pH, faecal        consistency, dry matter content and VFA concentrations. The only        clear effect of virginiamycin in this experiment was to reduce        the concentration of d-lactic acid. Virginiamycin had this        effect on the lactic acid at all levels of inclusion.

TABLE 9.2 Summary of faecal pH and lactic acid concentrations in dogsfed a diet of tinned “No Frills” dogs food and added raftilose. Aconsistency score of 5 = firm, normal faeces and 1 = diarrhoeaPre-Raftilose (mean all Control VM VM VM Treat dogs) No VM 0.25 0.5 1.0signif pH 6.0 5.4 5.2 4.9 4.9 Consistency 5.0 3.4 3.6 3.7 3.8 Dry matter25.0 20.5 22.0 22.5 24.2 L-lactic 0.6 11.5 2.2 13.8 11.8 D-lactic 0.18.3 0.2 0.4 0.3 ** Total VFA 88.0 80.0 80.5 93.7 73.5Discussion: There were high concentrations of VFA in all treatmentgroups irrespective of whether or not raftilose was included in thediet. It is likely that this high level of VFA has an overriding effecton pH and masks the potential beneficial effects of reduced D-lacticacid associated with the inclusion of virginiamycin. The observationthat virginiamycin reduced D-lactic acid but had to little effect onL-lactic confirms that virginiamycin does not always inhibit all lacticacid bacteria.2. Discovery and isolation of lactic acid bacteria from dogs

Two dogs were fed a commercial dry dog food for 4 weeks before and thenduring the experimental period. Virginiamycin (Eskalin 20) was added tothe diet of one dog for a period of 7 days prior to faecal sampling toprovide 0.5 mg/kg body weight. Samples of faeces were taken for theanalysis of pH and lactic acid concentrations at the same time as thosetaken for isolation of lactic acid producing bacteria.

Isolation and purification of the lactic acid bacteria was achievedusing the methods previously described in Example 1. Two lactic acidbacteria were isolated from the dog not treated with virginiamycin and afurther two from the dog treated with virginiamycin (see Table 9.3)

TABLE 9.3 Characteristics of faeces and details of the lactic acidproducing bacteria isolated from dogs. Control (no virginiamycin) “M”Virginiamycin “T” L(+)Lactate 2.55 mmol/L 0.34 mmol/L D(−)Lactate 2.06mmol/L 0.45 mmol/L Faecal pH 5.49 5.34 Lab code No. LAB 03/D35 LAB04/D37 LAB 05/D23 LAB 06/D29 Description Predominantly rods inPredominantly large Predominantly small Long > than 5 μm thickfilaments. (Gram+) cocci, occurring cocci < than 1 μm in rods, insingles and mainly in pairs and diameter mainly as short chains. singly(larger than diplococci (Gram+) the S. bovis isolates SB R1 (Gram+)Preliminary Prevotella-like Streptococcus-like Enterocossus-likeNon-slime producing classification lactic acid isolate Accession No.NM00/12632 NM00/12633 NM00/12634 NM00/12635 Despot date Jun. 29, 2000Jun. 29, 2000 Jun. 29, 2000 Jun. 29, 2000

An experiment was conducted to discover and isolate the most importantlactic acid producing bacteria from the large bowel of a human subjectin good health and consuming a normal balanced diet with a range ofdifferent sources of carbohydrate (bread, pasta, rice and potato),various sources of protein (plant and animal origin) and a range offruit and vegetables providing soluble and insoluble non-starchpolysaccharides. The methods used to isolate the most important lacticacid bacteria were as described in Example 1.

The pH of the faecal sample was 6.5 and there were very low levels oflactic acid (1.0 mmol/kg L(+) lactate). Descriptions of the two dominantlactic acid producing isolates are given in Table 9.4 together withdetails of the AGAL lodgement.

TABLE 9.4 Description of lactic acid producing bacterial isolates fromhuman faeces Lab code of lactic acid isolate LAB 07/H1 LAB 08/H15Description Predominantly short rods Endospore-forming forming filamentsbacteria, rod shaped, straight with spores. (Gram+) PreliminaryBacteriodes-like Non-slime producing classification lactic acid isolateAGAL accession No. NM00/12636 NM00/12637 Date of AGAL Jun. 29, 2000 Jun.29, 2000 deposition

Example 10 Isolation of Lactic Acid Producing Bacteria from the Stomach

While most lactic acid production as a result of fermentation ofcarbohydrate takes place in the large bowel or hind gut it is alsopossible for fermentation and acid accumulation to occur in the stomachof some vertebrate species. Acid accumulation in the stomach can beimportant in development of ulcers in the stomach. While acidic damageis typically considered to be a result of acid secreted by the stomach.It is generally considered that the true stomach of monogastric animalsand humans is too acidic (around pH 2) for growth and persistence ofmost bacterial species. It is certainly not considered that bacterialfermentation in the stomach contributes to the acid load in thatdigestive compartment. However there are non-acid secreting regions inthe stomach of most monogastric animal and it is in these parts thatlactic acid producing bacteria to survive. In some vertebrates,including the horse, feed can be held in the stomach for several hours.Under these conditions it is possible that bacteria capable of survivingacidic conditions while fermenting carbohydrate may contribute to theacid load in the stomach.

An experiment was conducted to determine if lactic acid bacteria occurthe stomach of a horse consuming grain. A horse was fed a dietconsisting of pasture and 2 kg/d of ground wheat grain for 2 weeksbefore it was humanely killed. Contents of the stomach were removed andsampled for isolation of dominant lactic acid producing bacteria usingmethods described in Example 1.

A mixed culture of lactic acid bacteria was discovered and isolated fromthe stomach contents. There were numerous colonies even at 10⁻⁸ dilutionand lactic acid production was predominantly L(+) lactate. Thepurification of individual strains has not yet been completed.

The results of this experiment show that lactic acid producing bacteriain the stomach of monogastric animals other than horses may have animportant role in contributing to the acid load in the stomach that isresponsible for ulcer formation in a wide range of intensively fed andmanaged domestic animals as well as humans. Isolation of these strains,using the methods described in Example 1, is likely to yield strains oflactic acid bacteria with important roles in vaccine preparation,diagnostic methods and developing pharmaceutical preparations.

Example 11 Vaccination of Horses with the Combination of S. bovis (Sb R1A2) and Streptococcus equinus (SE R2)

Seven horses grazing senescent winter pastures were treated againstinternal parasites using a broad spectrum ivermectin drench three weeksbefore the start of the experiment. At the start of the experiment threeof the horse were vaccinated against the lactic acid producing bacteria:S. bovis (strain Sb R1-A2) and S. equinus (strain SE R2). Three weeksafter the primary vaccination the secondary vaccination wasadministered. Samples of blood and faeces were taken prior tovaccination, at the time of the secondary vaccination and again 2 weekslater.

Vaccine preparation and procedure: Pure isolation of S. bovis and S.equinus were thawed and cultured in Basal Medium 10 at 37°. Formalin wasadded to the cultures at the end of the growth phase to achieve a finalconcentration of 0.5%. The cells were harvested by centrifugation at17,000 g for 20 minutes. The supernatant was discarded beforere-suspending the cells in sterile PBS followed by centrifugation. Thecells were washed 3 times in this way before being counted and made to afinal volume in sterile PBS containing 1×10¹⁰ cells/ml. Prior tovaccination equal volumes of each bacterial suspension were mixed. Themixed bacterial suspension was then combined with aluminum hydroxideadjuvant (Alhydrogel 1.3%) in equal volume. The vaccine was administeredsubcutaneously on the neck of each horse (2 ml per injection with 5×10⁹cells of each bacteria per injection). Following each injection horseswere regularly checked for any side effects of the vaccination and theinjection site monitored by measurement.Results: The site reaction to the vaccine was clearly observed in allhorses and decreased in volume to around 10 ml within 48 h for theprimary injection and 96 h for the secondary vaccination. No adverseside effects were observed during the period of monitoring followingvaccination.

TABLE 11.1 Site reaction (volume in ml of subcutaneous oedema) followingprimary and secondary vaccination into the neck of horses with the dualbacterin Sb A2 and SE R2 Primary Secondary vaccination vaccination HorseID 24 h 48 h 24 h 48 h 96 h 1 12 12 59 36 9 2 54 15 90 20 9 3 5 5 56 6 6Average: 23.7 10.7 68.2 20.5 8.0 SE 15.3 3.0 10.9 8.8 1.2

TABLE 11.2 Antibody levels (ELISA absorbance) against S. bovis (A2) andS. equinus in serum from vaccinated horses S. equinus (SE R2) S. bovis(A2) Pre- Pre- Week Week Horse ID vaccination Week 3 Week 5 vaccination3 5 1 0.099 0.087 0.110 0.053 0.053 0.087 2 0.097 0.086 0.135 0.0480.100 0.130 3 0.088 0.128 0.165 0.060 0.096 0.129 Average: 0.095 0.1000.137 0.054 0.083 0.115 SE 0.003 0.014 0.016 0.003 0.015 0.014

There was good seroconversion and consistent antibody production againstboth bacterins in all horses (Table 11.2). The seroconversion andantibody levels measured in serum are consistent with the site reactiondetailed in Table 11.1 and demonstrate efficacy of vaccination usingdual lactic acid bacterins.

Conclusion: Multiple bacterin vaccines against lactic acid bacteria areeffective in producing seroconversion and consistent antibody responsesto both antigens.

INDUSTRIAL APPLICABILITY

The present invention makes use of a vaccine for the prevention oflactic acidosis in a vertebrate, said vaccine comprising at least oneisolated microorganism, or fragment or fragments thereof, wherein saidmicroorganism is capable of producing lactic acid within the gut of saidvertebrate, and wherein said microorganism is selected from the groupconsisting of: Clostridium-like species, Prevotella-like species,Bacteroides-like species, Enterococcus-like species, Selenomonasspecies, non-dextran slime producing Streptococcus species and non-slimeproducing lactic acid bacterial isolates.

TABLE 1 Fermentation products of Streptococcus bovis, Streptococcusequinus, Clostridium-like vitulinus and Selenomonas ruminantium isolatesin a broth of basal medium 10 with glucose (0.5%). Isolate End productsStreptococcus bovis L-Lactic ⁺ Streptococcus aqueous L-LacticClostridium-like vitulinus D-Lactic (LV R1, LV Clostridium-likevitulinus L-Lactic, D-Lactic (LV R3) Selenomonas ruminantium L-LacticAcetic, Propionic (SR R1) Selenomonas ruminantium L-Lactic, D-Lactic,Acetic, (SR R2) Selenomonas ruminantium L-Lactic, D-Lactic, Acetic, (SRR3) ⁺Bold indicates main fermentation product.

TABLE 2 Fermentation of carbohydrate substrates by S. bovis, S. equinus,Clostridium-like vitulinus and S. ruminantium isolates frompasture-adapted sheep supplemented with wheat grain plus urea. BacterialIsolates Streptococcus Clostridium-like vitulinus S. riuninantiumCarbohydrate B. bovis S. equinus LV R1 LV R2 LV R3 LV R4 LV R5 SR R1 SRR2 SR R3 Arabinose − − − − − − − − − − Cellobiose + + + + − + + + + +Fructose + + + + + + + + + + Galactose + + − − + − − + + +Glucose + + + + + + + + + + Glycerol − − − − − − − − − − Inulin  +*− + + − + + − − − Lactose +  −** − − + − − + + + Maltose + + + +− + + + + + Mannitol − − − − + − − + − − Mannose + + + + + + + + + +Raffinose + + + + + + + + + + Ribose − − − − − − − + + − Sorbitol − − −− − − − + + − Starch + − − − − − − − + + Sucrose + + + + + + + + + +Xylose − − − − − − − − − + *Two of the white pigmented S. bovis isolatesdid not ferment inulin; **one S. equinus isolate germ on lactose.

TABLE 3 Lactate production (mmol/l) by S. ruminantium (SR R1, SR R2 andSR R3) cultures after 24 h of anaerobic incubation in a broth of basalmedium 10 with glucose or starch added at 0.5% a 39° C. Type A Type BIsolates → S. ruminantium S. ruminantim (SR R1) (SR R2 and SR R3)Carbohydrate Glucose Starch Glucose Starch L-Lactate 23.7 0.0 20.1 15.5D-Lactate 0.0 0.0 19.9 15.1 Total (mmol/l) 23.7 0.0 40.0 26.6 Initial pH6.91 7.20 6.91 7.20

TABLE 4 Lactate production (mmol/l) by S. bovis and Clostridium-likevitulinus cultures after 24 h of anaerobic incubation in a broth ofbasal medium 10 with glucose or starch added at 0.5% at 39° C. The pH ofBM 10 with glucose was 6.92 and with starch was 7.20. S. bovis* C-likevitulinus Isolates (Sb R1, Sb R2, (LV R1, LV R2, C-like vitulinus Carbo-Sb R3) LV R4, LV R5) (LV R3) hydrate Glucose Starch Glucose StarchGlucose Starch L-Lactate 41.1 42.2 0.0 0.0 20.7 0.0 D-Lactate 0.0 0.039.3 0.0 20.6 0.0 Total 41.1 42.8 39.3 0.0 41.3 0.0 (mmol/l) *Meanvalues ± SE.

TABLE 5 Effect of virginiamycin (VM) concentration on the growth ofdifferent bacterial isolates of S. bovis, Clostridium-like vitulinus andS. ruminantium in a broth of medium 10 with glucose added at 0.5%. VMlevel (μg/ml) Bacterial isolate 0 2 4 6 8 Incubation period (h) → 24 4824 48 24 48 24 48 24 48 S. bovis and S. equinus + + − − − − − − − −(orange pigmented) S. bovis and S. equinus + + − − − − − − − − (whitepigmented) Clostridium-like + + − + − + − + − + vitulinus (LV R1)Clostridium-like + + + + − + − − − − vitulinus (LV R2)Clostridium-like + + + + − + − + − + vitulinus (LV R3)Clostridium-like + + + + + + − + − + vitulinus (LV R4)Clostridium-like + + − + − + − + − − vitulinus (LV R5) S. ruminantium(SR R1, + + + + + + + + + + SR R2 and SR R3) *24-h incubation, **48+ hincubation.

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1. A vaccine comprising at least one isolated microorganism or living ordead cells thereof wherein the microorganism is selected from the groupconsisting of: (a) Streptococcus bovis strain SbR1 Accession numberNM99/04455, (b) Streptococcus equinus strain SER1 Accession number:NM99/04456, (c) Streptococcus equinus strain SER2 Accession number:NM99/04457, (d) Selenomonas ruminantium strain SRR1 Accession number:NM99/04458, (e) Selenomonas ruminantium strain SRR3 Accession number:NM99/04460, (f) Clostridium vitulinus strain LVR3 Accession number:NM99/04461, (g) Clostridium vitulinus strain LVR4 Accession number:NM99/04462, (h) Prevotella isolates LAB01 Accession number: NM00/12630,(i) Prevotella isolate LAB03 Accession number: NM00/12632, (j)Bacteroides isolates LAB07 Accession Number: NM00/12636, (k) Bacteroidesisolate LAB05 Accession number: NM00/12634, (l) non-dextran slimeproducing Streptococcus isolate LAB04 Accession number: NM00/12633, (m)non-slime producing lactic acid bacterial isolates LAB02 Accessionnumber: NM00/12631, (n) non-slime producing lactic acid bacterialisolate LAB06 Accession number: NM00/12635, and (o) non-slime producinglactic acid bacterial isolate LAB08 Accession number: NM00/12637.
 2. Thevaccine of claim 1, wherein said dead cells are intact cells.
 3. Thevaccine of claim 1, wherein the vaccine is formulated for administrationvia intramuscular, subcutaneous, or inhalation routes.
 4. Apharmaceutical composition comprising the vaccine composition of claim 1and a pharmaceutically acceptable carrier, adjuvant and/or diluent,wherein said pharmaceutical composition is effective for the preventionof lactic acidosis in said monogastric, herbivore, or ruminant animal.5. The pharmaceutical composition according to claim 4, furthercomprising at least one cytokine.
 6. A method for inducing an immuneresponse against lactic acidosis in a vertebrate, comprisingadministering to said vertebrate an immunologically effective amount ofthe pharmaceutical composition according to claim
 4. 7. A method for thetreatment and/or prophylaxis of lactic acidosis in a vertebrate in needof said treatment and/or prophylaxis, wherein said method comprisesadministering intramuscularly, subcutaneously, or via inhalation to saidvertebrate a therapeutically effective amount of a pharmaceuticalcomposition according to claim
 4. 8. A method for inducing an immuneresponse against lactic acidosis in a vertebrate, comprisingadministering intramuscularly, subcutaneously, or via inhalation to saidvertebrate an immunologically effective amount of the vaccine inaccordance with claim
 1. 9. The method according to claim 8, furthercomprising administering at least one cytokine.
 10. A method for thetreatment and/or prophylaxis of lactic acidosis in a vertebrate in needof said treatment and/or prophylaxis, wherein said method comprisesadministering intramuscularly, subcutaneously, or via inhalation to saidvertebrate a therapeutically effective amount of the vaccine inaccordance with claim
 1. 11. The method of claim 10, wherein said methodfurther comprises the administration of an active agent, wherein saidactive agent is selected from the group consisting of: antibiotics,enzyme preparations, clay preparations, compounds which slow the digestaflow, prebiotics and probiotics.
 12. An isolated culture of at least onemicroorganism selected from the group consisting of: (a) Streptococcusbovis strain SbR1 Accession number NM99/04455, (b) Streptococcus equinusstrain SER1 Accession number: NM99/04456, (c) Streptococcus equinusstrain SER2 Accession number: NM99/04457, (d) Selenomonas ruminantiumstrain SRR1 Accession number: NM99/04458, (e) Selenomonas ruminantiumstrain SRR3 Accession number: NM99/04460, (f) Clostridium vitulinusstrain LVR3 Accession number: NM99/04461, (g) Clostridium vitulinusstrain LVR4 Accession number: NM99/04462, (h) Prevotella isolates LAB01Accession number: NM00/12630, (i) Prevotella isolate LAB03 Accessionnumber: NM00/12632, (j) Bacteroides isolates LAB07 Accession Number:NM00/12636, (k) Bacteroides isolate LAB05 Accession number: NM00/12634,(l) non-dextran slime producing Streptococcus isolate LAB04 Accessionnumber: NM00/12633, (m) non-slime producing lactic acid bacterialisolates LAB02 Accession number: NM00/12631, (n) non-slime producinglactic acid bacterial isolate LAB06 Accession number: NM00/12635, and(o) non-slime producing lactic acid bacterial isolate LAB08 Accessionnumber: NM00/12637.